26,27-homologated-20-epi-2-alkyl-19-nor-vitamin D compounds

ABSTRACT

This invention provides a novel class of vitamin D related compounds, namely, the 2-alkyl-19-nor-vitamin D derivatives, as well as a general method for their chemical synthesis. The compounds have the formula:                    
     where Y 1  and Y 2 , which may be the same or different, are each selected from the group consisting of hydrogen and a hydroxy-protecting group, R 6  is selected from the group consisting of alkyl, hydroxyalkyl and fluoroalkyl, and where the group R represents any of the typical side chains known for vitamin D type compounds. These 2-substituted compounds, especially the 2α-methyl and the 2α-methyl-20S derivatives, are characterized by relatively high intestinal calcium transport activity and relatively high bone calcium mobilization activity resulting in novel therapeutic agents for the treatment of diseases where bone formation is desired, particularly low bone turnover osteoporosis. These compounds also exhibit pronounced activity in arresting the proliferation of undifferentiated cells and inducing their differentiation to the monocyte thus evidencing use as anti-cancer agents and for the treatment of diseases such as psoriasis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.09/541,470 filed Mar. 31, 2000 now U.S. Pat. No. 6,316,642, which inturn is a continuation-in-part of U.S. patent application Ser. No.09/454,013 filed Dec. 3, 1999 now U.S. Pat. No. 6,277,837, which in turnis a divisional of U.S. patent application Ser. No. 09/135,463 filedAug. 17, 1998 now U.S. Pat. No. 6,127,559, which in turn is acontinuation-in-part of U.S. patent application Ser. No. 08/819,694filed Mar. 17, 1997 now U.S. Pat. No. 5,945,410.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with United States government support awarded bythe following agencies:

NIH DK 14881-26S1

The United States has certain rights in this invention.

BACKGROUND OF THE INVENTION

This patent invention relates to vitamin D compounds, and moreparticularly to vitamin D derivatives substituted at the carbon 2position.

The natural hormone, 1α,25-dihydroxyvitamin D₃ and its analog inergosterol series, i.e. 1α,25-dihydroxyvitamin D₂ are known to be highlypotent regulators of calcium homeostasis in animals and humans, and morerecently their activity in cellular differentiation has beenestablished, Ostrem et al., Proc. Natl. Acad. Sci. USA, 84, 2610 (1987).Many structural analogs of these metabolites have been prepared andtested, including 1α-hydroxyvitamin D₃, 1α-hydroxyvitamin D₂, variousside chain homologated vitamins and fluorinated analogs. Some of thesecompounds exhibit an interesting separation of activities in celldifferentiation and calcium regulation. This difference in activity maybe useful in the treatment of a variety of diseases such as renalosteodystrophy, vitamin D-resistant rickets, osteoporosis, psoriasis,and certain malignancies.

Recently, a new class of vitamin D analogs has been discovered, i.e. theso called 19-nor-vitamin D compounds, which are characterized by thereplacement of the A-ring exocyclic methylene group (carbon 19), typicalof the vitamin D system, by two hydrogen atoms. Biological testing ofsuch 19-nor-analogs (e.g., 1α,25-dihydroxy-19-nor-vitamin D₃) revealed aselective activity profile with high potency in inducing cellulardifferentiation, and very low calcium mobilizing activity. Thus, thesecompounds are potentially useful as therapeutic agents for the treatmentof malignancies, or the treatment of various skin disorders. Twodifferent methods of synthesis of such 19-nor-vitamin D analogs havebeen described (Perlman et al., Tetrahedron Lett. 31, 1823 (1990);Perlman et al., Tetrahedron Lett. 32, 7663 (1991), and DeLuca et al.,U.S. Pat. No. 5,086,191).

In U.S. Pat. No. 4,666,634, 2β-hydroxy and alkoxy (e.g., ED-71) analogsof 1α,25-dihydroxyvitamin D₃ have been described and examined by Chugaigroup as potential drugs for osteoporosis and as antitumor agents. Seealso Okano et al., Biochem. Biophys. Res. Commun. 163 1444 (1989). Other2-substituted (with hydroxyalkyl, e.g., ED-120, and fluoroalkyl groups)A-ring analogs of 1α,25-dihydroxyvitamin D₃ have also been prepared andtested (Myamoto et al., Chem. Pharm. Bull. 41, 1111 (1993), Nishii etal., Osteoporosis Int. Suppl. 1, 190 (1993); Posner et al., J. Org.Chem. 59 7855 (1994), and J. Org. Chem. 60g, 4617 (1995)).

Recently, 2-substituted analogs of 1α,25-dihydroxy-19-norvitamin D₃ havealso been synthesized, i.e. compounds substituted at 2-position withhydroxy or alkoxy groups (DeLuca et al., U.S. Pat. No. 5,536,713), whichexhibit interesting and selective activity profiles. All these studiesindicate that binding sites in vitamin D receptors can accommodatedifferent substituents at C-2 in the synthesized vitamin D analogs.

In a continuing effort to explore the 19-nor class of pharmacologicallyimportant vitamin D compounds, their analogs which are characterized bythe presence of an alkyl (particularly methyl) substituent at the carbon2 (C-2), i.e. 2-alkyl-19-nor-vitamin D compounds, and particularly2-methyl-19-nor-vitamin D compounds, have now been synthesized andtested. Such vitamin D analogs seemed interesting targets because therelatively small alkyl (particularly methyl) group at C-2 should notinterfere with binding to the vitamin D receptor. On the other hand itis obvious that a change of conformation of the cyclohexanediol ring, Acan be expected for these new analogs.

BRIEFS SUMMARY OF THE INVENTION

A class of 1α-hydroxylated vitamin D compounds not known heretofore arethe 19-nor-vitamin D analogs having an alkyl (particularly methyl) groupat the 2-position, i.e. 2-alkyl-19-nor-vitamin D compounds, particularly2-methyl-19-nor-vitamin D compounds.

Structurally these novel analogs are characterized by the generalformula I shown below:

where Y₁ and Y₂, which may be the same or different, are each selectedfrom the group consisting of hydrogen and a hydroxy-protecting group, R₆is selected from the group consisting of alkyl, hydroxyalkyl andfluoroalkyl, and where the group R represents any of the typical sidechains known for vitamin D type compounds.

More specifically R can represent a saturated or unsaturated hydrocarbonradical of 1 to 35 carbons, that may be straight-chain, branched orcyclic and that may contain one or more additional substituents, such ashydroxy- or protected-hydroxy groups, fluoro, carbonyl, ester, epoxy,amino or other heteroatomic groups. Preferred side chains of this typeare represented by the structure below

where the stereochemical center (corresponding to C-20 in steroidnumbering) may have the R or S configuration, (i.e. either the naturalconfiguration about carbon 20 or the 20-epi configuration), and where Zis selected from Y, —OY, —CH₂OY, —C≡CY, —CH═CHY, and —CH₂CH₂CH═CR³R⁴,where the double bond may have the cis or trans geometry, and where Y isselected from hydrogen, methyl, —COR⁵ and a radical of the structure:

where m and n, independently, represent the integers from 0 to 5, whereR¹ is selected from hydrogen, deuterium, hydroxy, protected hydroxy,fluoro, trifluoromethyl, and C₁₋₅-alkyl, which may be straight chain orbranched and, optionally, bear a hydroxy or protected-hydroxysubstituent, and where each of R², R³, and R⁴, independently, isselected from deuterium, deuteroalkyl, hydrogen, fluoro, trifluoromethyland C₁₋₅ alkyl, which may be straight-chain or branched, and optionally,bear a hydroxy or protected-hydroxy substituent, and where R¹ and R²,taken together, represent an oxo group, or an “allylidene group, ═CR²R³,or the group —(CH₂)_(p)—, where p is an integer from 2 to 5, and whereR³ and R⁴, taken together, represent an oxo group, or the group—(CH₂)_(q)—, where q is an integer from 2 to 5, and where R⁵ representshydrogen, hydroxy, protected hydroxy, C₁₋₅ alkyl or —OR⁷ where R⁷represents C₁₋₅ alkyl, and wherein any of the CH-groups at positions 20,22, or 23 in the side chain may be replaced by a nitrogen atom, or whereany of the groups —CH(CH₃)—, —CH(R³)—, or —CH(R²)— at positions 20, 22,and 23, respectively, may be replaced by an oxygen or sulfur atom.

The wavy lines to the substituents at C-2 and at C-20 indicate that thecarbon 2 and carbon 20 may have either the R or S configuration.

Specific important examples of side chains with natural20R-configuration are the structures represented by formulas (a), b),(c), (d) and (e) below. i.e. the side chain as it occurs in25-hydroxyvitamin D₃ (a); vitamin D₃ (b); 25-hydroxyvitamin D₂ (c);vitamin D₂ (d); and the C-24 epimer of 25-hydroxyvitamin D₂ (e):

Specific important examples of side chains with the unnatural 20S (alsoreferred to as the 20-epi) configuration are the structures presented byformulas (f) and (g) below:

The above novel compounds exhibit a desired, and highly advantageous,pattern of biological activity. These compounds are characterized byrelatively high intestinal calcium transport activity, as compared tothat of 1α,25-dihydroxyvitamin D₃, while also exhibiting relatively highactivity, as compared to 1α,25-dihydroxyvitamin D₃, in their ability tomobilize calcium from bone. Hence, these compounds are highly specificin their calcemic activity. Their preferential activity on mobilizingcalcium from bone and either high or normal intestinal calcium transportactivity allows the in vivo administration of these compounds for thetreatment of metabolic bone diseases where bone loss is a major concern.Because of their preferential calcemic activity on bone, these compoundswould be preferred therapeutic agents for the treatment of diseaseswhere bone formation is desired, such as osteoporosis, especially lowbone turnover osteoporosis, steroid induced osteoporosis, senileosteoporosis or postmenopausal osteoporosis, as well as osteomalacia andrenal osteodystrophy. The treatment may be transdermal, oral orparenteral. The compounds may be present in a composition in an amountfrom about 0.1 μg/gm to about 50 μg/gm of the composition, and may beadministered in dosages of from about 0.9 μg/day to about 50 μg/day.

The compounds of the invention are also especially suited for treatmentand i prophylaxis of human disorders which are characterized by animbalance in the immune system, e.g. in autoimmune diseases, includingmultiple sclerosis, diabetes mellitus, host versus graft reaction, andrejection of transplants; and additionally for the treatment ofinflammatory diseases, such as rheumatoid arthritis and asthma, as wellas the improvement of bone fracture healing and improved bone grafts.Acne, alopecia, skin conditions such as dry skin (lack of dermalhydration), undue skin slackness (insufficient skin firmness),insufficient sebum secretion and wrinkles, and hypertension are otherconditions which may be treated with the compounds of the invention.

The above compounds are also characterized by high cell differentiationactivity. Thus, these compounds also provide therapeutic agents for thetreatment of psoriasis, or as an anti-cancer agent, especially againstleukemia, colon cancer, breast cancer and prostate cancer. The compoundsmay be present in a composition to treat psoriasis in an amount fromabout 0.01 μg/gm to about 100 μg/gm of the composition, and may beadministered topically, transdermally, orally or parenterally in dosagesof from about 0.01 μg/day to about 100 μg/day.

This invention also provides novel intermediate compounds formed duringthe synthesis of the end products.

This invention also provides a novel synthesis for the production of theend products of structure I.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating the relative activity of a mixture of 2αand 2β-methyl-19-nor-20S-1α,25-dihydroxyvitamin D₃, a mixture of 2α and2β-methyl-19-nor-1α,25-dihydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃to compete for binding of [³H]-1,25-(OH)₂-D₃ to the pig intestinalnuclear vitamin D receptor;

FIG. 2 is a graph illustrating the percent HL-60 cell differentiation asa function of the concentration of a mixture of 2α and2β-methyl-19-nor-20S-1α,25-dihydroxyvitamin D₃, a mixture of 2α and2β-methyl-19-nor-1,25-dihydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃;

FIG. 3 if a graph similar to FIG. 1 except illustrating the relativeactivity of the individual compounds 2α and2β-methyl-19-nor-20S-1α,25-dihydroxyvitamin D₃,2α and2,2-methyl-19-nor-1α,25-dihydroxyvitamin D₃ and 1α,25-dihydroxyvitaminD₃ to compete for binding of [³H]-1,25-(OH)₂-D₃ to the vitamin D pigintestinal nuclear receptor;

FIG. 4 is a graph similar to FIG. 2 except illustrating the percentHL-60 cell differentiation as a function of the concentration of theindividual compounds 2α and 2β-methyl-19-nor-20S-1α,25-dihydroxyvitaminD₃, 2α and 2β-methyl-19-nor-1α,25-dihydroxyvitamin D₃ and1α,25-dihydroxyvitamin D₃;

FIG. 5 is a graph illustrating the relative activity of the individualcompounds 2α and 2β-hydroxymethyl-19-nor-20S-1α,25-dihydroxyvitamin D₃,2α and 2β-hydroxymethyl-19-nor-1α,25-dihydroxyvitamin D₃ and1α,25-dihydroxyvitamin D₃ to complete for binding of [³H]-1,25-(OH)₂-D₃to the vitamin D pig intestinal nuclear receptor; and

FIG. 6 is a graph illustrating the percent HL-60 cell differentiation asa function of the concentration of the individual compounds 2α and2β-hydroxymethyl-19-nor-20S-1α,25-dihydroxyvitamin D₃, 2α and2β-hydroxymethyl-19-nor-1α,25-dihydroxyvitamin D₃ and1α,25-dihydroxyvitamin D₃.

DETAILED DESCRIPTION OF THE INVENTION

As used in the description and in the claims, the term“hydroxy-protecting group” signifies any group commonly used for thetemporary protection of hydroxy functions, such as for example,alkoxycarbonyl, acyl, alkylsilyl or alkylarylsilyl groups (hereinafterreferred to simply as “silyl” groups), and alkoxyalkyl groups.Alkoxycarbonyl protecting groups are alkyl-O—CO— groupings such asmethoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl,butoxycarbonyl, isobutoxycarbonyl, tert-butoxycarbonyl,benzyloxycarbonyl or allyloxycarbonyl. The term “acyl” signifies analkanoyl group of 1 to 6 carbons, in all of its isomeric forms, or acarboxyalkanoyl group of 1 to 6 carbons, such as an oxalyl, malonyl,succinyl, glutaryl group, or an aromatic acyl group such as benzoyl, ora halo, nitro or alkyl substituted benzoyl group. The word “alkyl” asused in the description or the claims, denotes a straight-chain orbranched alkyl radical of 1 to 10 carbons, in all its isomeric forms.Alkoxyalkyl protecting groups are groupings such as methoxymethyl,ethoxymethyl, methoxyethoxymethyl, or tetrahydrofuranyl andtetrahydropyranyl. Preferred silyl-protecting groups are trimethylsilyl,triethylsilyl, t-butyldimethylsilyl, dibutylmethylsilyl,diphenylmethylsilyl, phenyldimethylsilyl, diphenyl-t-butylsilyl andanalogous alkylated silyl radicals. The term “aryl” specifies a phenyl-,or an alkyl-, nitro- or halo-substituted phenyl group.

A “protected hydroxy” group is a hydroxy group derivatised or protectedby any of the above groups commonly used for the temporary or permanentprotection of hydroxy functions, e.g. the silyl, alkoxyalkyl, acyl oralkoxycarbonyl groups, as previously defined. The terms “hydroxyalkyl”,“deuteroalkyl” and “fluoroalkyl” refer to an alkyl radical substitutedby one or more hydroxy, deuterium or fluoro groups respectively.

It should be noted in this description that the term “24-homo” refers tothe addition of one methylene group and the term “24-dihomo” refers tothe addition of two methylene groups at the carbon 24 position in theside chain. Likewise, the term “trihomo” refers to the addition of threemethylene groups. Also, the term “26,27-dimethyl” refers to the additionof a methyl group at the carbon 26 and 27 positions so that for exampleR³ and R⁴ are ethyl groups. Likewise, the term “26,27-diethyl” refers tothe addition of an ethyl group at the 26 and 27 positions so that R³ andR⁴ are propyl groups.

In the following lists of compounds, the particular substituent attachedat the carbon 2 position should be added to the nomenclature. Forexample, if a methyl group is the alkyl substituent, the term “2-methyl”should precede each of the named compounds. If an ethyl group is thealkyl substituent, the term “2-ethyl” should precede each of the namedcompounds, and so on. In addition, if the methyl group attached at thecarbon 20 position is in its epi or unnatural configuration, the term“20(S)” or “20-epi” should be included in each of the following namedcompounds. Also, if the side chain contains an oxygen atom substitutedat any of positions 20, 22 or 23, the term “20-oxa”, 422-oxa” or“23-oxa”, respectively, should be added to the named compound. The namedcompounds could also be of the vitamin D₂ type if desired.

Specific and preferred examples of the 2-alkyl-compounds of structure Iwhen the side chain is unsaturated are:

19-nor-24-homo-1,25-dihydroxy-22,23-dehydrovitamin D₃;

19-nor-24-dihomo-1,25-dihydroxy-22,23-dehydrovitarin D₃;

19-nor-24-trihomo-1,25-dihydroxy-22,23-dehydrovitamin D₃;

19-nor-26,27-dimethyl-24-homo-1,25-dihydroxy-22,23-dehydrovitamin D₃;

19-nor-26,27-dimethyl-24-dihomo-1,25-dihydroxy-22,23-dehydrovitamin D₃;

19-nor-26,27-dimethyl-24-trihomo-1,25-dihydroxy-22,23-dehydrovitamin D₃;

19-nor-26,27-diethyl-24-homo-1,25-dihydroxy-22,23-dehydrovitamin D₃;

19-nor-26,27-diethyl-24-dihomo-1,25-dihydroxy-22,23-dehydrovitamin D₃;

19-nor-2627-diethyl-24-trihomo-1,25-dihydroxy-22,23-dehydrovitarin D₃;

19-nor-26,27-dipropoyl-24-homo-1,25-dihydroxy-22,23-dehydrovitamin D₃;

19-nor-26,27-dipropyl-24-dihomo-1,25-dihydroxy-22,23-dehydrovitamin D₃;and

19-nor-26,27-dipropyl-24-trihomo-1,25-dihydroxy-22,23-dehydrovitamin D₃.

With respect to the above unsaturated compounds, it should be noted thatthe double bond located between the 22 and 23 carbon atoms in the sidechain may be in either the (E) or (Z) configuration. Accordingly,depending upon the configuration, the term “22,23(E)” or “22,23(Z)”should be included in each of the above named compounds. Also, it iscommon to designate the double bond located between the 22 and 23 carbonatoms with the designation “Δ²²”. Thus, for example, the first namedcompound above could also be written as19-nor-24-homo-22,23(E)-Δ²²-1,25-(OH)₂D₃ where the double bond is in the(E) configuration. Similarly, if the methyl group attached at carbon 20is in the unnatural configuration, this compound could be written as19-nor-20(S)-24-homo-22,23)-Δ²²-1,25-(OH)₂D₃.

Specific and preferred examples of the 2-alkyl-compounds of structure Iwhen the side chain is saturated are:

19-nor-24-homo-1,25-dihydroxyvitamin D₃;

19-nor-24-dihomo-1,25-dihydroxyvitamin D₃;

19-nor-24-trihomo-1,25-dihydroxyvitamin D₃;

19-nor-26,27-dimethyl-24-homo-1,25-dihydroxyvitamin D₃;

19-nor-26,27-dimethyl-24-dihomo-1,25-dihydroxyvitamin D₃;

19-nor-26,27-dimethyl-24-trihomo-1,25-dihydroxyvitamin D₃;

19-nor-26,27-diethyl-24-homo-1,25-dihydroxyvitamin D₃;

19-nor-26,27-diethyl-24-dihomo-1,25-dihydroxyvitamin D₃;

19-nor-26,27-diethyl-24-trihomo-1,25-dihydroxyvitamin D₃;

19-nor-26,27-dipropyl-24-homo-1,25-dihydroxyvitamin D₃;

19-nor-26,27-dipropyl-24-dihomo-1,25-dihydroxyvitamin D₃; and

19-nor-26,27-dipropyl-24-trihomo-1,25-dihydroxyvitamin D₃.

As noted previously, the above saturated side chain compounds shouldhave the appropriate 2-alkyl substituent and/or carbon 20 configurationadded to the nomenclature. For example, particularly preferred compoundsare:

19-nor-26,27-dimethyl-20(S)-2α-methyl-1α,25-dihydroxyvitamin D₃; whichcan also be written as19-nor-26,27-dihomo-20(S)-2α-methyl-1α,25-dihydroxyvitamin D₃;

19-nor-26,27-dimethyl-20(S)-2β-methyl-1α,25-dihydroxyvitamin D₃; whichcan also be written as19-nor-26,27-dihomo-20(S)-2β-methyl-1α,25-dihydroxyvitamin D₃;

19-nor-26,27-dimethylene-20(S)-2α-methyl-1α,25-dihydroxyvitamin D₃; and

19-nor-26,27-dimethylene-20(S)-2β-methyl-1α,25-dihydroxyvitamin D₃.

The preparation of 1α-hydroxy-2-alkyl-19-nor-vitamin D compounds,particularly p-hydroxy-2-methyl-19-nor-vitamin D compounds, having thebasic structure I can be accomplished by a common general method, i.e.the condensation of a bicyclic Windaus-Grundmann type ketone II with theallylic phosphine oxide III to the corresponding2-methylene-19-nor-vitamin D analogs IV followed by a selectivereduction of the exomethylene group at C-2 in the latter compounds:

In the structures II, III, and IV groups Y, and Y₂ and R representgroups defined above; Y, and Y₂ are preferably hydroxy-protectinggroups, it being also understood that any functionalities in R thatmight be sensitive, or that interfere with the condensation reaction, besuitable protected as is well-known in the art. The process shown aboverepresents an application of the convergent synthesis concept, which hasbeen applied effectively for the preparation of vitamin D compounds[e.g. Lythgoe et al., J. Chem. Soc. Perkin Trans. I, 590 (1978);Lythgoe, Chem. Soc. Rev. 9, 449 (1983); Toh et al., J. Org. Chem. 4.81414 (1983); Baggiolini et al., J. Org. Chem. 51, 3098 (1986); Sardinaet al., J. Org. Chem. 51 1264 (1986); J. Org. Chem. Li, 1269 (1986);DeLuca et al., U.S. Pat. No. 5,086,191; DeLuca et al., U.S. Pat. No.5,536,713].

Hydrindanones of the general structure II are known, or can be preparedby known methods. Specific important examples of such known bicyclicketones are the structures with the side chains (a), (b), (c) and (d)described above, i.e. 25-hydroxy Grundmann's ketone (f) [Baggiolini etal., J. Org. Chem, 51,3098 (1986)]; Grundmann's ketone (g) [Inhoffen etal., Chem. Ber. 90 664 (1957)]; 25-hydroxy Windaus ketone (h)[Baggiolini et al., J. Org. Chem., 51, 3098 (1986)] and Windaus ketone(i) [Windaus et al., Ann., 524,297 (1936)]:

For the preparation of the required phosphine oxides of generalstructure III, a new synthetic route has been developed starting frommethyl quinicate derivative 1, easily obtained from commercial(1R,3R,4S,5R)-(−)-quinic acid as described by Perlman et al.,Tetrahedron Lett. 32 7663 (1991) and DeLuca et al., U.S. Pat. No.5,086,191. The overall process of transformation of the starting methylester 1 into the desired A-ring synthons, is summarized by the SCHEME I.Thus, the secondary 4-hydroxyl group of 1 was oxidized with RuO₄ (acatalytic method with RuCl₃ and NaIO₄ as co-oxidant). Use of such astrong oxidant was necessary for an effective oxidation process of thisvery hindered hydroxyl. However, other more commonly used oxidants canalso be applied (e.g. pyridinium dichromate), although the reactionsusually require much longer time for completion. Second step of thesynthesis comprises the Wittig reaction of the sterically hindered4-keto compound 2 with ylide prepared from methyltriphenylphosphoniumbromide and n-butyllithium. Other bases can be also used for thegeneration of the reactive methylenephosphorane, like t-BuOK, NaNH₂,NaH, K/HMPT, NaN(TMS)₂, etc. For the preparation of the 4-methylenecompound 3 some described modifications of the Wittig process can beused, e.g. reaction of 2 with activated methylenetriphenyl-phosphorane[Corey et al., Tetrahedron Lett. 26, 555 (1985)]. Alternatively, othermethods widely used for methylenation of unreactive ketones can beapplied, e.g. Wittig-Homer reaction with the PO-ylid obtained frommethyldiphenylphosphine oxide upon deprotonation with n-butyllithium[Schosse et al., Chimia 3, 197 (1976)], or reaction of ketone withsodium methylsulfinate [Corey et al., J. Org. Chem. 2, 1128 (1963)] andpotassium methylsulfinate [Greene et al., Tetrahedron Lett. 3755(1976)]. Reduction of the ester with lithium aluminum hydride or othersuitable reducing agent (e.g. DIBALH) provided the diol 4 which wassubsequently oxidized by sodium periodate to the cyclohexanonederivative 5. The next step of the process comprises the Petersonreaction of the ketone 5 with methyl(trimethylsilyl)acetate. Theresulting allylic ester 6 was treated with diisobutylaluminum hydrideand the formed allylic alcohol 7 was in turn transformed to the desiredA-ring phosphine oxide 8. Conversion of 7 to 8 involved 3 steps, namely,in situ tosylation with n-butyllithium and p-toluenesulfonyl chloride,followed by reaction with diphenylphosphine lithium salt and oxidationwith hydrogen peroxide.

Several 2-methylene-19-nor-vitamin D compounds of the general structureIV may be synthesized using the A-ring synthon 8 and the appropriateWindaus-Grundmann ketone II having the desired side chain structure.Thus, for example, Wittig-Horner coupling of the lithium phosphinoxycarbanion generated from 8 and n-butyllithium with the protected25-hydroxy Grundmann's ketone 9 prepared according to publishedprocedure [Sicinski et al., J. Med. Chem. 37, 3730 (1994)] gave theexpected protected vitamin compound 10. This, after deprotection with AG50W-X4 cation exchange resin afforded1α,25-dihydroxy-2-methylene-19-nor-vitamin D₃ (11).

The final step of the process was the selective homogeneous catalytichydrogenation of the exomethylene unit at carbon 2 in the vitamin 11performed efficiently in the presence oftris(triphenylphosphine)rhodium(I) chloride [Wilkinson's catalyst,(Ph₃P)₃RhCl]. Such reduction conditions allowed to reduce only C(2)=CH₂unit leaving C(5)-C(8) butadiene moiety unaffected. The isolatedmaterial is an epimeric mixture (ca. 1:1) of 2-methyl-19-nor-vitamins 12and 13 differing in configuration at C-2. The mixture can be usedwithout separation or, if desired, the individual 2α- and 2β-isomers canbe separated by an efficient HPLC system.

A similar chemoselectivity was also observed in hydroboration reactionsto synthesize 2-hydroxymethyl derivatives 20 and 21 (see Scheme III).For this purpose, 9-borabicyclo(3.3.1)nonane (9-BBN) was used as areagent and reaction conditions analogous as those used by Okamura forhydroboration of simple vitamin D compounds. See J. Org. Chem. 1978,43,1653-1656 and J. Org. Chem. 1977,42,2284-2291. Since this literatureprecedent concerned hydroboration of 1-desoxy compounds, namely, (5E)-and (5Z)-isomers of vitamin D₂ and D₃, the process was first testedusing 1α,25-(OH)₂D₃ as a model compound. The formed organoboraneintermediate was subsequently oxidized with basic hydrogen peroxide.Such hydroboration-oxidation conditions allowed the exclusivehydroxylation of the C(2)=CH₂ unit in the vitamin 11, leaving theintercyclic C(5)=C(6)-C(7)=C(8) diene moiety unaffected. The isolatedepimeric mixture of 2-hydroxymethyl derivatives 20 and 21 (ca. 1:2, 35%yield) was purified and separated by straight- and reversed-phase HPLC.

The C-20 epimerization was accomplished by the analogous coupling of thephosphine oxide 8 with protected 20(S)-25-hydroxy Grundmann's ketone 15(SCHEME II) and provided 19-nor-vitamin 16 which after hydrolysis of thehydroxy-protecting groups gave20(S)-1α,25-dihydroxy-2-methylene-19-nor-vitamin D₃ (17). Hydrogenationof 17 using Wilkinson's catalyst provided the expected mixture of the2-methyl-19-nor-vitamin D analogs 18 and 19. Subsequent hydroborationwith 9-BBN yielded 20(S)-2-hydroxymethyl derivatives 22 and 23 (seeScheme III).

As noted above, other 2-methyl-19-nor-vitamin D analogs may besynthesized by the method disclosed herein. For example,1α-hydroxy-2-methylene-19-nor-vitamin D₃ can be obtained by providingthe Grundmann's ketone (g); subsequent reduction of the A-ringexomethylene group in the formed compound can give the correspondingepimeric mixture of 1α-hydroxy-2-methyl-19-nor-vitamin D₃ compounds.

A number of oxa-analogs of vitamin D₃ and their synthesis are alsoknown. For example, 20-oxa analogs are described in N. Kubodera at al,Chem. Pharm. Bull., 34, 2286 (1986), and Abe et al, FEBS Lett. 222, 58,1987. Several 22-oxa analogs are described in E. Murayama et al, Chem.Pharm. Bull., 34,4410 (1986), Abe et al, FEBS Lett., 226, 58 (1987), PCTInternational Application No. WO 90/09991 and European PatentApplication, publication number 184 112, and a 23-oxa analog isdescribed in European Patent Application, publication number 78704, aswell as U.S. Pat. No. 4,772,433.

This invention is described by the following illustrative examples. Inthese. examples specific products identified by Arabic numerals (e.g. 1,2, 3, etc) refer to the specific structures so identified in thepreceding description and in the SCHEME I and SCHEME II.

EXAMPLE 1

Preparation of 1α,25-dihydroxy-2α- and1α,25-dihydroxy-2β-methyl-19-nor-vitamin D₃ (12 and 13).

Referring first to SCHEME I the starting methyl quinicate derivative 1was obtained from commercial (−)-quinic acid as described previously[Perlman et al., Tetrahedron Lett. 32, 7663 (1991) and DeLuca et al.,U.S. Pat. No. 5,086,191]. 1: mp. 82-82.5° C. (from hexane), ¹H NMR(CDCl₃) δ 0.098, 0.110, 0.142, and 0.159 (each 3H, each s, 4× SiCH₃),0.896 and 0.911 (9H and 9H, each s, 2× Si-t-Bu), 1.820 (1H, dd, J=13.1,10.3 Hz), 2.02 (1H, ddd, J=14.3, 4.3, 2.4 Hz), 2.09 (1H, dd, J=14.3, 2.8Hz), 2.19 (1H, ddd, J=13.1, 4.4, 2.4 Hz), 2.31 (1H, d, J=2.8 Hz, OH),3.42 (1H, m; after D₂O dd, J=8.6, 2.6 Hz), 3.77 (3H, s), 4.12 (1H, m),4.37 (1H, m), 4.53 (1H, br s, OH).

(a) Oxidation of 4-hydroxy Group in Methyl Quinicate Derivative 1.

(3R,5R)-3,5-Bis[(tert-butyldimethylsilyl)oxy]-1-hydroxy-4-oxocyclohexanecarboxylicAcid Methyl Ester (2). To a stirred mixture of ruthenium(III) chloridehydrate (434 mg, 2.1 mmol) and sodium periodate (10.8 g, 50.6 mmol) inwater (42 mL) was added a solution of methyl quinicate 1 (6.09 g, 14mmol) in CCl₄/CH₃CN (1:1, 64 mL). Vigorous stirring was continued for 8h. Few drops of 2-propanol were added, the mixture was poured into waterand extracted with chloroform. The organic extracts were combined,washed with water, dried (MgSO₄) and evaporated to give a dark oilyresidue (ca. 5 g) which was purified by flash chromatography. Elutionwith hexane/ethyl acetate (8:2) gave pure, oily 4-ketone 2 (3.4 g, 56%):¹H NMR (CDCl₃) δ 0.054, 0.091, 0.127, and 0.132 (each 3H, each s, 4×SiCH₃), 0.908 and 0.913 (9H and 9H, each s, 2× Si-t-Bu), 2.22 (1H, dd,J=13.2, 11.7 Hz), 2.28 (1H, dt, J=14.9, 3.6 Hz), 2.37 (1H, dd, J=14.9,3.2 Hz), 2.55 (1H, ddd, J=13.2, 6.4, 3.4 Hz), 3.79 (3H, s), 4.41 (1H, t,J˜3.5 Hz), 4.64 (1H, s, OH), 5.04 (1H, dd, J=11.7, 6.4 Hz); MS m/z(relative intensity) no M⁺, 375 (M⁺-t-Bu, 32), 357 (M⁺-t-Bu-H₂O, 47),243 (31), 225 (57), 73 (100).

(b) Wittig Reaction of the 4-ketone 2.

(3R,5R)-3,5-Bis[(tert-butyldimethylsilyl)oxy]-1-hydroxy-4-methylenecyclohexanecarboxylicAcid Methyl Ester (3). To the methyltriphenylphoshonium bromide (2.813g, 7.88 mmol) in anhydrous THF (32 mL) at 0° C. was added dropwisen-BuLi (2.5 M in hexanes, 6.0 mL, 15 mmol) under argon with stirring.Another portion of MePh₃P⁺Br (2.813 g, 7.88 mmol) was then added and thesolution was stirred at 0° C. for 10 min and at room temperature for 40min. The orange-red mixture was again cooled to 0° C. and a solution of4-ketone 2 (1.558 g, 3.6 mmol) in anhydrous THF (16+2 mL) was syphonedto reaction flask during 20 min. The reaction mixture was stirred at 0°C. for 1 h and at room temperature for 3 h. The mixture was thencarefully poured into brine cont. 1% HCl and extracted with ethylacetate and benzene. The combined organic extracts were washed withdiluted NaHCO₃ and brine, dried (MgSO₄) and evaporated to give an orangeoily residue (ca. 2.6 g) which was purified by flash chromatography.Elution with hexane/ethyl acetate (9:1) gave pure 4-methylene compound 3as a colorless oil (368 mg, 24%): ¹H NMR (CDCl₃) δ 0.078, 0.083, 0.092,and 0.115 (each 3H, each s, 4× SiCH₃), 0.889 and 0.920 (9H and 9H, eachs, 2× Si-t-Bu), 1.811 (1H, dd, J=12.6, 11.2 Hz), 2.10 (2H, m), 2.31 (1H,dd, J=12.6, 5.1 Hz), 3.76 (3H, s), 4.69 (1H, t, J=3.1 Hz), 4.78 (1H, m),4.96 (2H, m; after D₂OH, br s), 5.17 (1H, t, J=1.9 Hz); MS m/z (relativeintensity) no M⁺, 373 (M⁺-t-Bu, 57), 355 (M⁺-t-Bu-H₂O, 13), 341 (19),313 (25), 241 (33), 223 (37), 209 (56), 73 (100).

(c) Reduction of Ester Group in the 4-methylene Compound 3.

[(3′R,5′R)-3′,5′-Bis[(tert-butyldimethylsilyl)oxy]-1-hydroxy-4′-methylenecyclohexyl]methanol(4). (i) To a stirred solution of the ester 3 (90 mg, 0.21 mmol) inanhydrous THF (8 mL) lithium aluminum hydride (60 mg, 1.6 mmol) wasadded at 0° C. under argon. The cooling bath was removed after 1 h andthe stirring was continued at 6° C. for 12 h and at room temperature for6 h. The excess of the reagent was decomposed with saturated aq. Na₂SO₄,and the mixture was extracted with ethyl acetate and ether, dried(MgSO₄) and evaporated. Flash chromatography of the residue withhexanelethyl acetate (9:1) afforded unreacted substrate (12 mg) and apure, crystalline diol 4 (35 mg, 48% based on recovered ester 3): ¹H NMR(CDCl₃+D₂O) δ 0.079, 0.091, 0.100, and 0.121 (each 3H, each s, 4×SiCH₃), 0.895 and 0.927 (9H and 9H, each s, 2× Si-t-Bu), 1.339 (1H, t,J˜12 Hz), 1.510 (1H, dd, J=14.3, 2.7 Hz), 2.10 (2H, m), 3.29 and 3.40(1H and 1H, each d, J=11.0 Hz), 4.66 (1H, t, J˜2.8 Hz), 4.78 (1H, m),4.92 (1H, t, J=1.7 Hz), 5.13 (1H, t, J=2.0 Hz); MS m/z (relativeintensity) no M⁺, 345 (M⁺-t-Bu, 8), 327 (M⁺-t-Bu-H₂O, 22), 213 (28), 195(11), 73 (100). (ii) Diisobutylaluminum hydride (1.5 M in toluene, 2.0mL, 3 mmol) was added to a solution of the ester 3 (215 mg, 0.5 mmol) inanhydrous ether (3 mL) at −78° C. under argon. The mixture was stirredat −78° C. for 3 h and at −24° C. for 1.5 h, diluted with ether (10 mL)and quenched by the slow addition of 2N potassium sodium tartrate. Thesolution was warmed to room temperature and stirred for 15 min, thenpoured into brine and extracted with ethyl acetate and ether. Theorganic extracts were combined, washed with diluted (ca. 1%) HCl, andbrine, dried (MgSO₄) and evaporated. The crystalline residue waspurified by flash chromatography. Elution with hexane/ethyl acetate(9:1) gave crystalline diol 4 (43 mg, 24%).

(d) Cleavage of the Vicinal Diol 4.

(3R,5R)-3,5-Bis[(tert-butyldimethylsilyl)oxy]-4-methylenecyclohexanone(5). Sodium periodate saturated water (2.2 mL) was added to a solutionof the diol 4 (146 mg, 0.36 mmol) in methanol (9 mL) at 0° C. Thesolution was stirred at 0° C. for 1 h, poured into brine and extractedwith ether and benzene. The organic extracts were combined, washed withbrine, dried (MgSO₄) and evaporated. An oily residue was dissolved inhexane (1 mL) and applied on a silica Sep-Pak cartridge. Pure4-methylenecyclohexanone derivative 5 (110 mg, 82%) was eluted withhexane/ethyl acetate (95:5) as a colorless oil: ¹H NMR (CDCl₃) δ 0.050and 0.069 (6H and 6H, each s, 4× SiCH₃), 0.881 (18H, s, 2× Si-t-Bu),2.45 (2H, ddd, J=14.2, 6.9, 1.4 Hz), 2.64 (2H, ddd, J=14.2, 4.6, 1.4Hz), 4.69 (2H, dd, J=6.9, 4.6 Hz), 5.16 (2H, s); MS m/z (relativeintensity) no M⁺, 355 (M⁺-Me, 3), 313 (M⁺-t-Bu, 100), 73 (76).

(e) Preparation of the Allylic Ester 6.

[(3′R,5′R)-3′,5′-Bis[(tert-butyldimethylsilyl)oxy]-4′-methylenecyclohexylidene]aceticAcid Methyl Ester (6). To a solution of diisopropylamine (37 μL, 0.28mmol) in anhydrous THEF (200 μL) was added n-BuLi (2.5 M in hexanes, 113μL, 0.28 mmol) under argon at −78° C. with stirring, andmethyl(trimethylsilyl)acetate (46 μL, 0.28 mmol) was then added. After15 min, the keto compound 5 (49 mg, 0.132 mmol) in anhydrous THF (200+80μL) was added dropwise. The solution was stirred at −78° C. for 2 h andthe reaction mixture was quenched with saturated NH₄Cl, poured intobrine and extracted with ether and benzene. The combined organicextracts were washed with brine, dried (MgSO₄) and evaporated. Theresidue was dissolved in hexane (1 mL) and applied on a silica Sep-Pakcartridge. Elution with hexane and hexane/ethyl acetate (98:2) gave apure allylic ester 6 (50 mg, 89%) as a colorless oil: ¹H NMR (CDCl₃) δ0.039, 0.064, and 0.076 (6H, 3H, and 3H, each s, 4× SiCH₃), 0.864 and0.884 (9H and 9H, each s, 2× Si-t-Bu), 2.26 (1H, dd, J=12.8, 7.4 Hz),2.47 (1H, dd, J=12.8, 4.2 Hz), 2.98 (1H, dd, J=13.3, 4.0 Hz), 3.06 (1H,dd, 3=13.3, 6.6 Hz), 3.69 (3H, s), 4.48 (2H, m), 4.99 (2H, s), 5.74 (1H,s); MS m/z (relative intensity) 426 (M⁺, 2), 411 (M⁺-Me, 4), 369(M⁺-t-Bu, 100), 263 (69).

(f) Reduction of the Allylic Ester 6.

2-[(3′R,5′R)-3′,5′-Bis[(tert-butyldimethylsilyl)oxy]-4′-methylenecyclohexylidene]ethanol (7). Diisobutylaluminum hydride (1.5 M in toluene, 1.6 mL, 2.4mmol) was slowly added to a stirred solution of the allylic ester 6 (143mg, 0.33 mmol) in toluene/methylene chloride (2:1, 5.7 mL) at −78° C.under argon. Stirring was continued at −78° C. for 1 h and at −46° C.(cyclohexanone/dry ice bath) for 25 min. The mixture was quenched by theslow addition of potassium sodium tartrate (2N, 3 mL), aq. HCl (2N, 3mL) and H₂O (12 mL), and then diluted with methylene chloride (12 mL)and extracted with ether and benzene. The organic extracts werecombined, washed with diluted (ca. 1%) HCl, and brine, dried (MgSO₄) andevaporated. The residue was purified by flash chromatography. Elutionwith hexane/ethyl acetate (9:1) gave crystalline allylic alcohol 7 (130mg, 97%): ¹H NMR (CDCl₃) δ 0.038, 0.050, and 0.075 (3H, 3H, and 6H, eachs, 4× SiCH₃), 0.876 and 0.904 (9H and 9H, each s, 2× Si-t-Bu), 2.12 (1H,dd, J=12.3, 8.8 Hz), 2.23 (1H, dd, J=13.3, 2.7 Hz), 2.45 (1H, dd,J=12.3, 4.8 Hz), 2.51 (1H, dd, J=13.3, 5.4 Hz), 4.04 (1H, m; after D₂Odd, J=12.0, 7.0 Hz), 4.17 (1H, m; after D₂O dd, J=12.0, 7.4 Hz), 4.38(1H, m), 4.49 (1H, m), 4.95 (1H, br s), 5.05 (1H, t, J=1.7 Hz), 5.69(1H, t, J=7.2 Hz); MS m/z (relative intensity) 398 (M⁺, 2), 383 (M⁺-Me,2), 365 (M⁺-Me-H₂O, 4), 341 (M⁺-t-Bu, 78), 323 (M⁺-t-Bu-H₂O, 10), 73(100); exact mass calcd for C₂₇H₄₄O₃ 416.3290, found 416.3279.

(g) Conversion of the Allylic Alcohol 7 into Phosphine Oxide 8.

[2-[(3′R,5′R)-3′,5′-Bis[(tert-butyldimethylsilyl)oxy]-4′-methylenecyclohexylidene]ethyl]diphenylphosphineOxide (8). To the allylic alcohol 7 (105 mg, 0.263 mmol) in anhydrousTHF (2.4 mL) was added n-BuLi (2.5 M in hexanes, 105 μL, 0.263 mmol)under-argon at 0° C. Freshly recrystallized tosyl chloride (50.4 mg,0.264 mmol) was dissolved in anhydrous THE (480 μL) and added to theallylic alcohol-BuLi solution. The mixture was stirred at 0° C. for 5min and set aside at 0° C. In another dry flask with air replaced byargon, n-BuLi (2.5 M in hexanes, 210 μL, 0.525 mmol) was added to Ph₂PH(93 μL, 0.534 mmol) in anhydrous THF (750 μL) at 0° C. with stirring.The red solution was syphoned under argon pressure to the solution oftosylate until the orange color persisted (ca. 112 of the solution wasadded). The resulting mixture was stirred an additional 30 min at 0° C.,and quenched by addition of H₂O (30 μl). Solvents were evaporated underreduced pressure and the residue was redissolved in methylene chloride(2.4 mL) and stirred with 10% H₂O₂ at 0° C. for 1 h. The organic layerwas separated, washed with cold aq. sodium sulfite and H₂O, dried(MgSO₄) and evaporated. The residue was subjected to flashchromatography. Elution with benzenelethyl acetate (6:4) gavesemicrystalline phosphine oxide 8 (134 mg, 87%): ¹H NMR (CDCl₃) δ 0.002,0.011, and 0.019 (3H, 3H, and 6H, each s, 4× SiCH₃), 0.855 and 0.860 (9Hand 9H, each s, 2× Si-t-Bu), 2.0-2.1 (3H, br m), 2.34 (1H, m), 3.08 (1H,m), 3.19 (1H, m), 4.34 (2H, m), 4.90 and 4.94 (1H and 1H, each s,), 5.35(1H, ˜q, J=7.4 Hz), 7.46 (4H, m), 7.52 (2H, m), 7.72 (4H, m); MS-m/z(relative intensity) no M⁺, 581 (M⁺-1,1), 567 (M⁺-Me, 3), 525 (M⁺-t-Bu,100), 450 (10), 393 (48).

(h) Wittig-Homer Coupling of Protected 25-hydroxy Grundmann's Ketone 9with the Phosphine Oxide 8.

1α,25-Dihydroxy-2-methylene-19-nor-vitamin D₃ (11). To a solution ofphosphine oxide 8 (33.1 mg, 56.8 μmol) in anhydrous THB (450 μL) at 0°C. was slowly added n-BuLi (2.5 M in hexanes, 23 μL, 57.5 μmol) underargon with stirring. The solution turned deep orange. The mixture wascooled to −78° C. and a precooled (−78° C.) solution of protectedhydroxy ketone 9 (9.0 mg, 22.8 μmol), prepared according to publishedprocedure [Sicinski et al., S. Med. Chem. 37,3730 (1994)], in anhydrousTHF (200+100 μL) was slowly added. The mixture was stirred under argonat −78° C. for 1 h and at 0° C. for 18 h. Ethyl acetate was added, andthe organic phase was washed with brine, dried (MgSO₄) and evaporated.The residue was dissolved in hexane and applied on a silica Sep-Pakcartridge, and washed with hexane/ethyl acetate (99:1, 20 mL) to give19-nor-vitamin derivative 10 (13.5 mg, 78%). The Sep-Pak was then washedwith hexane/ethyl acetate (96:4, 10 mL) to recover some unchangedC,D-ring ketone 9 (2 mg), and with ethyl acetate (10 mL) to recoverdiphenylphosphine oxide (20 mg). For analytical purpose a sample ofprotected vitamin 10 was further purified by HPLC (6.2 mm×25 cmZorbax-Sil column, 4 mL/min) using hexane/ethyl acetate (99.9:0.1)solvent system. Pure compound 10 was eluted at R_(V) 26 mL as acolorless oil: UV (in hexane) λ_(max) 244, 253, 263 nm; ¹H NMR (CDCl₃) δ0.025, 0.049, 0.066, and 0.080 (each 3H, each s, 4× SiCH₃), 0.546 (3H,s, 18-H₃), 0.565 (6H, q, J=7.9 Hz, 3× SiCH₂), 0.864 and 0.896 (9H and9H, each s, 2× Si-t-Bu), 0.931 (3H, d, J=6.0 Hz, 21-H₃), 0.947 (9H, t,J=7.9 Hz, 3× SiCH₂CH₃), 1.188 (6H, s,26- and 27-H₃), 2.00 (2H, m), 2.18(1H, dd, J=12.5, 8.5 Hz, 4β-1H), 2.33 (1H, dd, J=13.1, 2.9 Hz, 10β-H),2.46 (1H, dd, J=12.5, 4.5 Hz, 4α-H), 2.52 (1H, dd, J=13.1, 5.8 Hz,10α-H), 2.82 (1H, br d, J=12 Hz, 90-H), 4.43 (2H, m, 1β- and 3α-H), 4.92and 4.97(1H and 1H, each s, ═CH₂), 5.84 and 6.22 (1H and 1H, each d,J=11.0 Hz, 7- and 6-H); MS m/z (relative intensity) 758 (M⁺, 17), 729(M⁺-Et, 6), 701 (M⁺-t-Bu, 4), 626 (100), 494 (23), 366 (50),73 (92).

Protected vitamin 10 (4.3 mg) was dissolved in benzene (150 μL) and theresin (AG 50W-X4, 60 mg; prewashed with methanol) in methanol (800 μL)was added. The mixture was stirred at room temperature under argon for17 h, diluted with ethyl acetate/ether (1:1, 4 mL) and decanted. Theresin was washed with ether (8 mL) and the combined organic phaseswashed with brine and saturated NaHCO₃, dried (MgSO₄) and evaporated.The residue was purified by HPLC (6.2 mm×25 cm Zorbax-Sil column, 4mL/min) using hexane/2-propanol (9:1) solvent system. Analytically pure2-methylene-19-nor-vitamin 11 (2.3 mg, 97%) was collected at R_(V) 29 mL(1α,25-dihydroxyvitamin D₃ was eluted at R_(v) 52 mL in the same system)as a white solid: UV (in EtOH) λ_(max) 243.5, 252, 262.5 nm; ¹H NMR(CDCl₃) δ 0.552 (3H, s, 18-H₃), 0.941 (3H, d, J=6.4 Hz, 21-H₃), 1.222(6H, s, 26- and 27-H₃), 2.01 (2H, m), 2.27-2.36 (2H, m), 2.58 (1H, m),2.80-2.88 (2H, m), 4.49 (2H, m, 1β- and 3α-H), 5.10 and 5.11 (1H and 1H,each s, ═CH₂), 5.89 and 6.37 (1H and 1H, each d, J=11.3 Hz, 7- and 6-H);MS m/z (relative intensity) 416 (M⁺, 83), 398 (25), 384 (31), 380 (14),351 (20), 313 (100); exact mass calcd for C₂₇H₄₄O₃ 416.3290, found416.3279.

(i) Hydrogenation of 2-methylene-19-nor-vitamin 11.

1α,25-Dihydroxy-2α- and 1α,25-Dihydroxy-2β-methyl-19-nor-vitamin D₃ (12and 13). Tris(triphenylphosphine)rhodium(I) chloride (2.3 mg, 2.5 μmol)was added to dry benzene (2.5 ml) presaturated with hydrogen. Themixture was stirred at room temperature until a homogeneous solution wasformed (ca. 45 min). A solution of vitamin 11 (1.0 mg, 2.4 μmol) in drybenzene (0.5 mL) was then added and the reaction was allowed to proceedunder a continuous stream of hydrogen for 3 h. Benzene was removed undervacuum, and hexane/ethyl acetate (1:1, 2 mL) was added to the residue.The mixture was applied on a silica Sep-Pak and both 2-methyl vitaminswere eluted with the same solvent system (20 mL). Further purificationwas achieved by HPLC (6.2 mm×25 cm Zorbax-Sil column, 4 mL/min) usinghexane/2-propanol (9:1) as a solvent system. The mixture (ca. 1:1) of2-methyl-19-nor-vitamins (2α- and 2β-epimers 12 and 13; 0.80 mg, 80%)gave a single peak at R_(v) 33 mL.

12 and 13: UV (in EtOH) λ_(max) 243, 251, 261.5 nm; ¹H NMR (CDCl₃) δ0.536 and 0.548 (3H and 3H, each s, 2×18-H₃), 0.937 (6H, d, J=6.3 Hz,2×21-H₃), 1.133 and 1.144 (3H and 3H, each d, J˜6 Hz, 2×2-CH₃), 1.219[12H, s, 2× (26- and 27-H₃)], 2.60 (1H, dd, J=13.0, 4.6 Hz), 2.80 (3H,m), 3.08 (1H, dd, J=12.6, 4.0 Hz), 3.51 (1H, dt, J=4.6, 10.2 Hz), 3.61(1H, dt, J=4.5, 9.1 Hz), 3.90 (1H, narr m), 3.96 (1H, narr m), 5.82,5.87, 6.26, and 6.37 (each 1H, each d, J=11.2 Hz); MS m/z (relativeintensity) 418 (M, 100), 400 (25), 385 (15), 289 (30), 245 (25).

Separation of both epimers was achieved by reversed-phase HPLC (10 mm×25cm Zorbax-ODS column, 4 mL/min) using methanol/water (85:15) solventsystem. 2β-Methyl vitamin 13 (0.35 mg, 35%) was collected at R_(v) 41 mLand its 2α-epimer 12 (0.34 mg, 34%) at R_(v) 46 mL.

12: UV (in EtOH) λ_(max) 243, 251, 261 nm; ¹H NMR (CDCl₃) δ 0.536 (3H,s, 18-H₃), 0.937 (3H, d, J=6.4 Hz, 21-H₃), 1.134 (3H, d, J=6.9 Hz,2α-CH₃), 1.218 (6H, s, 26- and 27-H₃), 2.13 (1H, -t, J˜12 Hz, 40-H),2.22 (1H, br d, J=13 Hz, 10β-H), 2.60 (1H, dd, J=12.8,4.3 Hz, 4α-H),2.80 (2H, m, 9β- and 10α-H), 3.61 (1H, m, w/2=24 Hz, 3α-H), 3.96 (1H, m,w/2=12 Hz, 1β-H), 5.82 and 6.37 (1H and 1H, each d, J=11.1 Hz, 7- and6-H); MS m/z (relative intensity) 418 (M⁺, 62), 400 (32), 385 (17), 289(36), 271 (17), 253 (20), 245 (43), 69 (100), 59 (74) ); exact masscalcd for C₂₇H₄₆O₃ 418.3447, found 418.3441.

13: UV (in EtOH) λ_(max) 242, 250.5, 261 nm; 1H NMR (CDCl₃) δ 0.548 (3H,s, 18-H₃), 0.940 (3H, d, J=6.4 Hz, 21-H₃), 1.143 (3H, d, J=6.8 Hz,2,-CH₃), 1.220 (6H, s, 26- and 27-H₃), 2.34 (1H, dd, J=13.7, 3.3 Hz,4β-H), 2.43 (1H, br d, J=13.7 Hz, 4α-H), 2.80 (1H, dd, J=12 and 4 Hz,9-H), 3.08 (1H, dd, J=13.0, 4.2 Hz, 10β-H), 3.51 (1H, m, w/2=25 Hz,1β-H), 3.90 (1H, m, w/2=11 Hz, 3α-H), 5.87 and 6.26 (1H and 1H, each d,J=11.2 Hz, 7- and 6-H); MS m/z (relative intensity) 418 (M⁺, 63), 400(47), 385 (16), 289 (40), 271 (32), 253 (27), 245 (47), 69 (100), 59(64); exact mass calcd for C₂₇H₄₆O₃ 418.3447, found 418.3436.

EXAMPLE 2

Preparation of 20(S)-1α,25-dihydroxy-2α- and20(S)-1α,25-dihydroxy-2,-methyl-19-nor-vitamin D₃ (18 and 19).

SCHEME II illustrates the preparation of protected 20(S)-25-hydroxyGrundmann's ketone 15, its coupling with phosphine oxide 8 (obtained asdescribed in Example 1) and selective hydrogenation of exomethylenegroup in 2-methylene compound 17.

(a) Silylation of Hydroxy Ketone 14.

20(S)-25-[(Triethylsilyl)oxy]-des-A,B-cholestan-8-one (15). A solutionof the ketone 14 (Tetrionics, Inc.; 56 mg, 0.2 mmol) and imidazole (65mg, 0.95 mmol) in anhydrous DMF (1.2 μL) was treated with triethylsilylchloride (95 μL, 0.56 mmol), and the mixture was stirred at roomtemperature under argon for 4 h. Ethyl acetate was added and water, andthe organic layer was separated. The ethyl acetate layer was washed withwater and brine, dried (MgSO₄) and evaporated. The residue was passedthrough a silica Sep-Pak cartridge in hexane/ethyl acetate (9:1), andafter evaporation, purified by HPLC (9.4 mm×25 cm Zorbax-Sil column, 4mL/min) using hexane/ethyl acetate (9:1) solvent system. Pure protectedhydroxy ketone 15 (55 mg, 70%) was eluted at R_(v) 35 mL as a colorlessoil: ¹H NMR (CDCl₃) δ 0.566 (6H, q, J=7.9 Hz, 3× SiCH₂), 0.638 (3H, s,18-H₃), 0.859 (3H, d, J=6.0 Hz, 21-H₃), 0.947 (9H, t, J=7.9 Hz, 3×SiCH₂CH₃), 1.196 (6H, s,26- and 27-H₃), 2.45 (1H, dd, J=11.4, 7.5 Hz,14α-H).

(b) Wittig-Horner Coupling of Protected 20(S)-25-hydroxy Grundmann'sKetone With the Phosphine Oxide 8.

20(S)-1α,25-Dihydroxy-2-methylene-19-nor-vitamin D₃ (17). To a solutionof phosphine oxide 8 (15.8 mg, 27.1 μmol) in anhydrous THF (200 μL) at0° C. was slowly added n-BuLi (2.5 M in hexanes, 11 μL, 27.5 μmol) underargon with stirring. The solution turned deep orange. The mixture wascooled to −78° C. and a precooled (−78° C.) solution of protectedhydroxy ketone 15 (8.0 mg, 20.3 μmol) in anhydrous THF (100 μL) wasslowly added. The mixture was stirred under argon at −78° C. for 1 h andat 0° C. for 18 h. Ethyl acetate was added, and the organic phase waswashed with brine, dried (MgSO₄) and evaporated. The residue wasdissolved in hexane and applied on a silica Sep-Pak cartridge, andwashed with hexane/ethyl acetate (99.5:0.5,20 mL) to give 19-nor-vitaminderivative 16 (7 mg, 45%) as a colorless oil. The Sep-Pak was thenwashed with hexane/ethyl acetate (96:4, 10 mL) to recover some unchangedC,D-ring ketone 15 (4 mg), and with ethyl acetate (10 mL) to recoverdiphenylphosphine oxide (9 mg). For analytical purpose a sample ofprotected vitamin 16 was further purified by BPLC (6.2 mm×25 cmZorbax-Sil column, 4 mL/min) using hexane/ethyl acetate (99.9:0.1)solvent system.

16: UV (in hexane) λ_(max) 244, 253.5, 263 nm; ¹H NMR (CDCl₃) δ 0.026,0.049, 0.066, and 0.080 (each 3H, each s, 4× SiCH₃), 0.541 (3H, s,18-H₃), 0.564 (6H, q, J=7.9 Hz, 3× SiCH₂), 0.848 (3H, d, J=6.5 Hz,21-H₃), 0.864 and 0.896 (9H and 9H, each s, 2× Si-t-Bu), 0.945 (9H, t,J=7.9 Hz, 3× SiCH₂CH₃), 1.188 (6H, s, 26- and 27-H₃), 2.15-2.35 (4H, brm), 2.43-2.53 (3H, br m), 2.82 (1H, br d, J=12.9 Hz, 9β-H), 4.42 (2H, m,1β- and 3α-H), 4.92 and 4.97 (11H and 11H, each s, ═CH₂), 5.84 and 6.22(1H and 1H, each d, 3=11.1 Hz, 7- and 6-H); MS m/z (relative intensity)758 (M⁺, 33), 729 (M⁺-Et, 7), 701 (M⁺-t-Bu, 5), 626 (100), 494 (25), 366(52), 75 (82), 73 (69).

Protected vitamin 16 (5.0 mg) was dissolved in benzene (160 μL) and theresin (AG 50W-X4, 70 mg; prewashed with methanol) in methanol (900 μL)was added. The mixture was stirred at room temperature under argon for19 h, diluted with ethyl acetate/ether (1:1, 4 mL) and decanted. Theresin was washed with ether (8 mL) and the combined organic phaseswashed with brine and saturated NaHCO₃, dried (MgSO₄) and evaporated.The residue was purified by HPLC (6.2 mm×25 cm Zorbax-Sil column, 4mL/min) using hexane/2-propanol (9:1) solvent system. Analytically pure2-methylene-19-nor-vitamin 17 (2.6 mg, 95%) was collected at R_(v) 28 mL[(20R)-analog was eluted at R_(v) 29 mL and 1α,25-dihydroxyvitamin D₃ atR_(v) 52 mL in the same system] as a white solid: WV (in EtOH) λ_(max)243.5, 252.5, 262.5 nm; 1H NMR (CDCl₃) δ 0.551 (3H, s, 18-H₃), 0.858(3H, d, J=6.6 Hz, 21-H₃), 1.215 (6H, s, 26- and 27-H₃), 1.95-2.04 (2H,m), 2.27-2.35 (2H, m), 2.58 (1H, dd, J=13.3, 3.7 Hz), 2.80-2.87 (2H, m),4.49 (2H, m, 1β- and 3α-H), 5.09 and 5.11 (1H and 1H, each s, ═CH₂),5.89 and 6.36 (1H and 1H, each d, J=11.3 Hz, 7- and 6-11); MS m/z(relative intensity) 416 (M⁺, 100), 398 (26), 380 (13), 366 (21), 313(31); exact mass calcd for C₂₇H₄₄O₃ 416.3290, found 416.3275.

(c) Hydrogenation of 2-methylene-19-nor-vitamin 17.

20(S)-1α,25-Dihydroxy-2α- and20(S)-1α,25-Dihydroxy-2β-methyl-19-nor-vitamin D₃ (18 and 19).Tris(triphenylphosphine)rhodium(l) chloride (2.3 mg, 2.5 μmol) was addedto dry benzene (2.5 mL) presaturated with hydrogen. The mixture wasstirred at room temperature until a homogeneous solution was formed (ca.45 min). A solution of vitamin 17 (1.0 mg, 2.4 μmol) in dry benzene (0.5mL) was then added and the reaction was allowed to proceed under acontinuous stream of hydrogen for 3 h. Benzene was removed under vacuum,and hexane/ethyl acetate (1:1, 2 mL) was added to the residue. Themixture was applied on a silica Sep-Pak and both 2-methyl vitamins wereeluted with the same solvent system (20 mL). Further purification wasachieved by HPLC (6.2 mm×25 cm Zorbax-Sil column, 4 mL/min) usinghexane/2-propanol (9:1) as a solvent system. The mixture (ca. 1:1) of2-methyl-19-nor-vitamins (2α- and 2β-epimers 18 and 19; 0.43 mg, 43%)gave a single peak at R_(v) 31 mL.

18 and 19: WV (in EtOH) λ_(max) 243, 251, 261 nm; ¹H NMR (CDCl₃) δ 0.534and 0.546 (3H and 3H, each s, 2×18-H₃), 0.852 and 0.857 (3H and 3H, eachd, J=6.5 Hz, 2×21-H₃), 1.133 (3H, d, J=6.7 Hz, 2-CH₃), 1.143 (3H, d,J=6.5 Hz, 2-CH₃), 1.214 [12H, s, 2× (26- and 27-H₃)], 2.60 (1H, dd,J=12.7, 4.5 Hz), 2.80 (3H, m), 3.08 (1H, dd, J=13.1, 4.3 Hz), 3.51 (1H,br m; after D₂0 dt, J=4.5, 10.0 Hz), 3.61 (1H, br m; after D₂O dt,J=4.4, 9.2 Hz), 3.90 (1H, narr m), 3.96 (1H, narr m), 5.82, 5.87, 6.26,and 6.37 (each 1H, each d, J=11.3 Hz); MS in/z (relative intensity) 418(M⁺, 100), 400 (45), 385 (20), 289 (38), 245 (47).

Separation of both epimers was achieved by reversed-phase HPLC (10 mm×25cm Zorbax-ODS column, 4 mL/min) using methanol/water (85:15) solventsystem. 2β-Methyl vitamin 19 (16%) was collected at R_(V) 36 mL and its2α-epimer 18 (20%) at R_(V) 45 mL.

18: WV (in EtOH) λ_(max) 242.5, 251, 261 nm; ¹H NMR (CDCl₃) δ 0.534 (3H,S, 18-H₃), 0.852 (3H, d, J=6.6 Hz, 21-H₃), 1.133 (3H, d, J=6.9 Hz,2α-CH₃), 1.214 (6H, s, 26- and 27-H₃), 2.13 (1H, ˜t, J˜12 Hz, 4β-1H),2.22 (1H, br d, J=13 Hz, 10β-H), 2.60 (1H, dd, J=12.8, 4.4 Hz, 4β-H),2.80 (2H, m, 9β- and 10α-H), 3.61 (1H, m, w/2=25 Hz, 3α-H), 3.95 (1H, m,w/2=11 Hz, 1β-H), 5.82 and 6.37 (1H and 1H, each d, J=11.2 Hz, 7- and6-H); MS m/z (relative intensity) 418 (M⁺, 58), 400 (25), 385 (20), 289(28), 271 (23), 253 (22), 245 (38), 69 (100), 59 (47) ); exact masscalcd for C₂₇H₄₆O₃ 418.3447, found 418.3450.

19: UV (in EtOH) λ_(max) 242.5, 250.5, 261 nm; ¹H NMR (CDCl₃) δ 0.547(3H, s, 18-H₃), 0.857 (3H, d, J=6.6 Hz, 21-H₃), 1.143 (3H, d, J=6.8 Hz,2β-CH₃), 1.214 (6H, s, 26- and 27-H₃), 2.34 (1H, dd, J=13.8, 3.1 Hz,413-H), 2.43 (1H,.br d, J=13.8 Hz, 4α-H), 2.80 (1H, dd, J=12 and 4 Hz,9β-H), 3.08 (1H, dd, J=12.9, 4.4 Hz, 10β-H), 3.50 (1H, m, w/2=26 Hz,1,-H), 3.89 (1H, m, w/2=11 Hz, 3α-H), 5.86 and 6.26 (1H and 1H, each d,J=11.2 Hz, 7- and 6-H); MS m/z (relative intensity) 418 (M⁺, 68), 400(47), 385 (21),289 (33), 271 (27), 253 (26), 245 (47), 69 (100), 59(53); exact mass calcd for C₂₇H₄₆O₃ 418.3447, found 418.3448.

1α,25-Dihydroxy-2α- and 1α,25-Dihydroxy-2β-(hydroxymethyl)-19-norvitaminD₃ (20 and 21). 9-Borabicyclo[3.3.1]nonane (0.5 M in THF, 60 μL, 30μmol) was added to a solution of vitamin 11 (1.25 mg, 3 μmol) inanhydrous THF (50 μL) at room temperature (evolution of hydrogen wasobserved). After 3 h of stirring, the mixture was quenched with methanol(20 μL), stirred for 15 min at room temperature, cooled to 0° C., andtreated successively with 6 M NaOH (10 μL, 60 μmol) and 30% H₂O₂ (10μL). The mixture was heated for 1 h at 55° C., cooled, benzene and brinewere added, and the organic phase was separated, dried and evaporated.The crystalline residue was dissolved in ether (0.5 mL) and kept infreezer overnight. The ether solution was carefully removed from theprecipitated crystals of cyclooctanediol and evaporated. Separation ofthe residue was achieved by HPLC (6.2 mm×25 cm Zorbax-Sil column, 4mL/min) using hexane/2-propanol (85:15) solvent system. Traces ofunreacted substrate 11 were eluted at R_(V) 16 mL, whereas isomeric2-hydroxymethyl vitamins 20 and 21 were collected at R_(V) 33 mL and 40mL, respectively. Further purification of both products byreversed-phase HPLC (10 mm×25 cm Zorbax-ODS column, 4 mL/min) usingmethanol/water (9:1) solvent system afforded analytically pure vitamin20 (0.14 mg, 11%) and its 2β-isomer 21 (0.31 mg, 24%) collected at R_(V)26 mL and 23 mL, respectively.

20: UV (in EtOH) λ_(max) 242.5, 250.5, 261 nm; ¹H NMR (CDCl₃) δ 0.536(3H, s, 18-H₃), 0.939 (3H, d, J=6.4 Hz, 21-H₃), 1.214 (6H, s, 26- and27-H₃), 2.13 (1H, br d, J=13.5 Hz, 10-H), 2.21 (1H, ˜t, J=12 Hz, 4β-H),2.64 (1H, dd, J=12.7, 4.5 Hz, 4α-H), 2.80 (2H, br d, J=12.7 Hz, 9β-H),2.90 (1H, br d, J=13.5 Hz, 10α-H), 3.95-4.1 (3H, br m, 2α-CH₂OH and3α-H), 4.23 (1H, m, w/2=11 Hz, 1β-H), 5.79 and 6.41 (1H and 1H, each d,J=11.1 Hz, 7- and 6-H); MS m/z (relative intensity) 434 (M⁺, 37), 416(33), 398 (16), 383 (10), 305 (12), 287 (25), 269 (26), 245 (40), 69(100), 59 (74); exact mass calcd for C₂₇H₄₆O₄ 434.3396, found 434.3397.

21: UV (in EtOH) λ_(max) 242, 250.5, 260.5 nm; ¹H NMR (CDCl₃) δ 0.553(3H, s, 18-H₃), 0.942 (3H, d, J=6.5 Hz, 21-H₃), 1.220 (6H, s, 26- and27-H₃), 2.31 (1H, br d, J=14 Hz, 4β-H), 2.45 (1H, br d, J=14 Hz, 4α-H),2.79 (1H, br d, J=13 Hz, 9-H), 3.17 (1H, dd, J=12.8,4.2 Hz, 10β-H),3.95-4.1 (3H, br m, 1β-H and 2β-CH₂OH), 4.17 (1H, m, w/2=10 Hz, 3α-H),5.89 and 6.26 (1H and 1H, each d, J=11.0 Hz, 7- and 6-H); MS m/z(relative intensity) 434 (M⁺, 50), 416 (46), 398 (20), 383 (11), 305(14), 287 (26), 269 (30), 245 (50), 69 (100), 59 (75); exact mass calcdfor C₂₇H₄₆O₄ 434.3396, found 434.3402.

20(S)-1α,25-Dihydroxy-2α- and20(S)-1α,25-Dihydroxy-2β-(hydroxymethyl)-19-norvitamin D₃ (22 and 23).The hydroboration of 20(S)-vitamin 17 and subsequent oxidation of theorganoborane adduct were performed using the procedure analogous to thatdescribed above for (20R)-epimer 11. The reaction products wereseparated by HPLC (6.2 mm×25 cm Zorbax-Sil column, 4 mL/min) usinghexane/2-propanol (87.5:12.5) solvent system, and the isomeric2-hydroxymethyl vitamins 22 and 23 were collected at R_(V) 40 mL and 47mL, respectively. Further purification of both products byreversed-phase HPLC (10 mm×25 cm Zorbax-ODS column, 4 mL/min) usingmethanol/water (9:1) solvent system afforded analytically pure vitamin22 (9%) and its 2β-isomer 23 (26%) collected at R_(V) 25 mL and 22 mL,respectively.

22: UV (in EtOH) λ_(max) 242.5, 250.5, 261 nm; ¹H NMR (CDCl₃) δ 0.532(3H, s, 18-H₃), 0.853 (3H, d, J=6.6 Hz, 21-H₃), 1.214 (6H, s, 26- and27-H₃), 2.13 (1H, br d, J=13.3 Hz, 10β-H), 2.21 (1H, t, J=12 Hz, 4β-H),2.64 (1H, dd, J=12.8, 4.3 Hz, 4α-H), 2.80 (1H, br d, J=12 Hz, 913-H),2.90 (1H, br d, J=13.3 Hz, 10α-H), 3.95-4.1 (3H, br m, 2α-CH₂OH and3α-H), 4.24 (1H, m, w/2=10 Hz, 1β-H), 5.81 and 6.43 (1H and 1H, each d,J=11.1 Hz, 7- and 6-H); MS m/z (relative intensity) 434 (M⁺, 41), 416(34), 398 (16), 383 (10), 305 (10), 287 (28), 269 (26), 245 (51), 69(100), 59 (82); exact mass calcd for C₂₇H₄₆O₄ 434.3396, found 434.3390.

23: UV (in EtOH) λ_(max) 242, 250.5, 260.5 nm; ¹H NMR (CDCl₃) δ 0.551(3H, s, 18-H₃), 0.861 (3H, d, J=6.4 Hz, 21-H₃), 1.215 (6H, s, 26- and27-H₃), 2.31 (1H, br d, J=13.7 Hz, 413-H), 2.45 (1H, br d, J=13.7 Hz,4α-H), 2.80 (1H, br d, J=12.5 Hz, 9β-H, 3.17 (1H, dd, J=12.7,4.2 Hz,10β-H), 3.95-4.1 (3H, br m, 1β-H and 2β-CH₂OH), 4.17 (1H, m, w/2=10 Hz,3α-H), 5.89 and 6.26 (1H and 1H, each d, J=11.1 Hz, 7- and 6-H); MS m/z(relative intensity) 434 (, 35), 416 (29), 398 (15), 383 (8), 305 (10),287 (18), 269 (23), 245 (48), 69 (100), 59 (93); exact mass calcd forC₂₇H₄₆O₄ 434.3396, found 434.3408.

Biological Activity of 2-methyl-Substituted 19-nor-1,25-(OH)₂D₃Compounds and Their 20S-Isomers

The synthesized 2-substituted vitamins were tested for their ability tobind the porcine intestinal vitamin D receptor (See FIGS. 1, 3 and 5). Acomparison between the natural hormone 1α,25-(OH)₂D₃ and 2-methylsubstituted 19-norvitamins 12, 18 and 19 shows that they are about asactive as 1α,25-(OH)₂D₃, while the 2β-methyl isomer in the 20R-series 13is 39-fold less effective. The 2α-hydroxymethyl vitamin D analog 22 withthe “unnatural” configuration at C-20 was almost equivalent to1α,25-(OH)₂D₃ with respect to receptor binding, and the isomeric 23proved to be less potent (6-8×) than these compounds. The corresponding2α-hydroxymethyl analog possessing the “natural” 20R-configuration 20exhibited about the same binding affinity as 23, whereas the 2β-isomer21 was ca. 8 times less effective. The foregoing results of thecompetitive binding analysis show that vitamins with the axialorientation of the 1α-hydroxy group exhibit a significantly enhancedaffinity for the receptor.

It might be expected from these results that all of these compoundswould have equivalent biological activity. Surprisingly, however, the2-methyl substitutions produced highly selective analogs with theirprimary action on bone. When given for 7 days in a chronic mode, themost potent compounds tested were a mixture of the α and β isomers of2-methyl 19-nor-20S-1,25-(OH)₂D₃ (Table 1). When given at 130 μmol/day,the activity of this mixture of compounds on bone calcium mobilization(serum calcium) was much higher than that of the native hormone,possibly as high as 10 or 100 times higher. Under identical conditions,twice the dose of 1,25-(OH)₂D₃ gave a serum calcium value of 7.2 mg/100ml, while a mixture of 2-methyl-(α and β)-19-nor-20S-1,25-(OH)₂D₃ gave avalue of 9.6 mg/100 ml of serum calcium at the 130 μmol dose. When givenat 260 μmol/day, this mixture produced the astounding value of 12.2mg/100 ml of serum calcium at the expense of bone. To show itsselectivity, these compounds produced no significant change inintestinal calcium transport at 130 μmol dose level while having astrong bone calcium mobilizing activity. At the higher dose, the2-methyl-20S mixture did produce an intestinal transport response butgave an enormous bone mobilization response. A mixture of the α and βisomers of 2-methyl-19-nor-1,25-(OH)₂D₃ also had strong bone calciummobilization at both dose levels but also showed no intestinal calciumtransport activity. Thus, the 2-methyl-α and β derivatives given as amixture showed strong preferential bone calcium mobilizing activityespecially when the side chain was in the 20S-configuration. Theseresults illustrate that the 2-methyl and the 20S-2-methyl derivatives of19-nor-1,25-(OH)₂D₃ are selective for the mobilization of calcium frombone. Table 2 illustrates the response of both intestine and serumcalcium to a single large dose of the various compounds; again,supporting the conclusions derived from Table 1.

The results in FIG. 2 illustrate that a mixture of the α and βderivatives of 2-methyl-19-nor-20S-1,25-(OH)₂D₃ is extremely potent ininducing differentiation of HL-60 cells to the moncyte. The 2-methyl-αand β compounds had activity similar to 1,25-(OH)₂D₃. These resultsillustrate the potential of the 2-methyl-19-nor-20S-1,25-(OH)₂D₃compounds as anti-cancer agents, especially against leukemia, coloncancer, breast cancer and prostate cancer, or as agents in the treatmentof psoriasis.

Competitive binding of the analogs to the porcine intestinal receptorwas carried out by the method described by Dame et al (Biochemnistry 25,4523-4534, 1986).

The differentiation of HL-60 promyelocytic into monocytes was determinedas described by Ostrem et al (J. Biol. Chem. 262, 14164-14171, 1987).

TABLE 1 Response of Intestinal Calcium Transport and Serum Calcium (BoneCalcium Mobilization) Activity to Chronic Doses of 2-Methyl Derivativesof 19-Nor-1,25-(OH)₂D₃ and its 20S Isomers Intestinal Calcium DoseTransport Serum Calcium Group (pmol/day/7 days) (S/M) (mg/100 ml)Vitamin D Deficient Vehicle 5.5 ± 0.2  5.1 ± 0.16 1,25-(OH)₂D₃ Treated260 6.2 ± 0.4 7.2 ± 0.5 2-Methyl (α and β) 130 5.0 ± 0.3 6.1 ± 0.119-Nor-1,25-(OH)₂D₃ 260 5.3 ± 0.6 6.7 ± 0.4 2-Methyl (α and β) 130 5.0 ±0.9 9.6 ± 0.1 19-Nor-20S-1,25-(OH)₂ 260 6.9 ± 0.5 12.2 ± 0.3  D₃

Male weanling rats were obtained from Sprague Dawley Co. (Indianapolis,Ind.) and fed a 0.47% calcium, 0.3% phosphorus vitamin D-deficient dietfor 1 week and then given the same diet containing 0.02% calcium, 0.3%phosphorus for 2 weeks. During the last week they were given theindicated dose of compound by intraperitoneal injection in 0.1 ml 95%propylene glycol and 5% ethanol each day for 7 days. The control animalsreceived only the 0.11 ml of 95% propylene glycol, 5% ethanol.Twenty-four hours after the last dose, the rats were sacrificed andintestinal calcium transport was determined by everted sac technique aspreviously described and serum calcium determined by atomic absorptionspectrometry on a model 3110 Perkin Elmer instrument Norwalk, Conn.).There were 5 rats per group and the values represent mean±SEM.

TABLE 2 Response of Intestinal Calcium Transport and Serum Calcium (BoneCalcium Mobilization) Activity to a Single Dose of the2-Methyl-Derivatives of 19-Nor-1,25-(OH)₂D₃ and its 20S IsomersIntestinal Calcium Transport Serum Calcium Group (S/M) (mg/100 ml) -DControl 4.2 ± 0.3 4.7 ± 0.1 1,25-(OH)₂D₃ 5.8 ± 0.3 5.7 ± 0.2 2-Methyl (αand β mixture)-19-Nor- 3.6 ± 0.4 5.4 ± 0.1 1,25-(OH)₂D₃ 2-Methyl (α andβ mixture)-19-Nor- 6.7 ± 0.6 8.1 ± 0.3 20S-1,25-(OH)₂D₃

Male Holtzman strain weanling rats were obtained from the Sprague DawleyCo. (Indianapolis, Ind.) and fed the 0.47% calcium, 0.3% phosphorus dietdescribed by Suda et al. (J. Nutr. 100 1049-1052, 1970) for 1 week andthen fed the same diet containing 0.02% calcium and 0.3% phosphorus for2 additional weeks. At this point, they received a single intrajugularinjection of the indicated dose dissolved in 0.1 ml of 95% propyleneglycol/5% ethanol. Twenty-four hours later they were sacrificed andintestinal calcium transport and serum calcium were determined asdescribed in Table 1. The dose of the compounds was 650 μmol and therewere 5 animals per group. The data are expressed as mean±SEM.

When given for 7 days in a chronic mode, the most potent individualcompound tested was 2α-methyl 19-nor-20S-1,25-(OH)₂D₃ (Table 3). Whengiven at 130 μmol/day, the activity of this compound on bone calciummobilization (serum calcium) was much higher than that of the nativehormone, possibly as high as 10 or 100 times higher. Under identicalconditions, twice the dose of 1,25-(OH)₂D₃ gave a serum calcium value of6.6±0.4 mg/100 ml, while 2α-methyl-19-nor-20S-1,25-(OH)₂D₃ gave a valueof 8.3±0.7 mg/100 ml of serum calcium at the 130 μmol dose. When givenat 260 μmol/day, 2α-methyl-19-nor-20S-1,25-(OH)₂D₃ produced theastounding value of 10.3±0.11 mg/100 ml of serum calcium at the expenseof bone. To show its selectivity, this compound also produced asignificant change in intestinal calcium transport at both the 260 μmoland the 130 μmol dose levels while having a strong bone calciummobilizing activity. At the higher dose, the 2α-methyl-20S compound didproduce a significant intestinal transport response but also gave anenormous bone mobilization response. With respect to the2β-methyl-19-nor-20S compound, the data in Table 3 show it has little,if any, intestinal calcium transport activity, and little, if-any, bonemobilization activity. The data in Table 4 illustrate that2α-methyl-19-nor-1,25-(OH)₂D₃ also had relatively strong bone calciummobilization at both dose levels and also showed some intestinal calciumtransport activity. In contrast, 2β-methyl-19-nor-1,25-(OH)₂D₃ showedlittle, if any, intestinal calcium transport or bone calciummobilization activities. Thus, the 2α-methyl-19-nor derivative showedstrong preferential bone calcium mobilizing activity especially when theside chain was in the 208-configuration. These results illustrate thatthe 2α-methyl and the 20S-2α-methyl derivatives of 19-nor-1,25-(OH)₂D₃are selective for the mobilization of calcium from bone.

The results in FIG. 4 illustrate that 2α-methyl-19-nor-20S-1,25-(OH)₂D₃and 2α-methyl-19-nor-1,25-(OH)₂D₃ are extremely potent in inducingdifferentiation of HL-60 cells to the monocyte. The 2β-methyl compoundshad activity similar to 1,25-(OH)₂D₃. These results illustrate thepotential of the 2α-methyl-19-nor-20S-1,25-(OH)₂D₃ compound as ananti-cancer agent, especially against leukemia, colon cancer, breastcancer and prostate cancer, or as an agent in the treatment ofpsoriasis.

Competitive binding of the analogs to the porcine intestinal receptorwas carried out by the method described by Dame et al (Biochemistry 25,4523-4534, 1986).

The differentiation of HL-60 promyelocytic into monocytes was determinedas described by Ostrem et al (J. Biol. Chem. 262, 14164-14171, 1987).

TABLE 3 Response of Intestinal Calcium Transport and Serum Calcium (BoneCalcium Mobilization) Activity to Chronic Doses of the 20S Isomers of2-Methyl Derivatives of 19-Nor-1,25-(OH)₂D₃ Intestinal Dose Calcium(pmol/ Transport Serum Calcium Group day/7 days) (S/M) (mg/100 ml)Vitamin D Deficient Vehicle 2.9 ± 0.2 4.2 ± 0.1 1,25-(OH)₂D₃ Treated 2604.6 ± 0.2 6.6 ± 0.4 2α-Methyl-19-nor-20(S)- 130 12.9 ± 1.9  8.3 ± 0.71,25-(OH)₂D₃ 260 8.4 ± 1.1 10.3 ± 0.11 2β-Methyl-19-nor-20(S)- 130 2.9 ±0.3 4.4 ± 0.1 1,25-(OH)₂D₃ 260 3.8 ± 0.1 4.4 ± 0.1

With respect to the data in Tables 3 and 4, male weanling rats wereobtained from Sprague Dawley Co. (Indianapolis, Ind.) and fed a 0.47%calcium, 0.3% phosphorus vitamin D-deficient diet for 1 week and thengiven the same diet containing 0.02% calcium, 0.3% phosphorus for 2weeks. During the last week they were given the indicated dose ofcompound by intraperitoneal injection in 0.1 ml 95% propylene glycol and5% ethanol each day for 7 days. The control animals received only the0.1 ml of 95% propylene glycol, 5% ethanol. Twenty-four hours after thelast dose, the rats were sacrificed and intestinal calcium transport wasdetermined by everted sac technique as previously described and serumcalcium determined by atomic absorption spectrometry on a model 3110Perkin Elmer instrument (Norwalk, Conn.). There were 5 rats per groupand the values represent mean±SEM.

TABLE 4 Response of Intestinal Calcium Transport and Serum Calcium (BoneCalcium Mobilization) Activity to Chronic Doses of the 2-MethylDerivatives of 19-Nor-1,25-(OH)₂D₃ Intestinal Calcium Dose TransportSerum Calcium Group Pmol/day/7 days (S/M) (mg/100 ml) -D Control  0 2.3± 0.8 3.9 ± 0.2 1,25-(OH)₂D₃ 260 5.6 ± 1.3 6.1 ± 0.5 2α-Methyl-19-nor-130 4.3 ± 1.0 4.8 ± 0.2 1,25-(OH)₂D₃ 260 5.3 ± 1.3 5.8 ± 0.52β-Methyl-19-nor- 130 4.4 ± 0.8 4.1 ± 0.1 1,25-(OH)₂D₃ 260 3.1 ± 0.9 3.8± 0.2

Table 5 provides intestinal calcium transport and bone calciummobilization data for 2-hydroxymethyl derivatives of19-nor-1α,25-(OH)₂D₃. These derivatives turned out to be relativelyinactive, including those in the 20S-series 22 and 23.

TABLE 5 Response of Intestinal Calcium Transport and Serum Calcium (BoneCalcium Mobilization) Activity to Chronic Doses of the2-Hydroxymethyl-Derivatives of 19-Nor-1,25-(OH)₂D₃ Intestinal CalciumDose Transport Serum Calcium Group Pmol/day/7 days (S/M) (mg/100 ml)Vitamin D Deficient Vehicle 4.0 ± 0.3 3.8 ± 0.1 1,25-(OH)₂D₃ Treated 2606.6 ± 0.5 5.2 ± 0.1 2α-Hydroxymethyl-19- 130 5.0 ± 0.3 4.0 ± 0.1nor-20(S)-1,25-(OH)₂D₃ 260 5.8 ± 0.4 3.9 ± 0.1 2β-Hydroxymethyl-19- 1303.5 ± 0.7 3.6 ± 0.1 nor-20(S)-1,25-(OH)₂D₃ 260 3.5 ± 0.3 3.5 ± 0.2

In the next assay, the cellular activity of the synthesized compoundswas established by studying their ability to induce differentiation ofhuman promyelocyte HL-60 cells into monocytes. It was found that all ofthe synthesized vitamin D analogs with the “unnatural” 20S-configurationwere more potent than 1α,25-(OH)₂D₃. Moreover, the same relationshipbetween cellular activity and conformation of the vitamin D compoundswas established as in the case of receptor binding analysis and in vivostudies, i.e. 2α-substituted vitamin D analogs were considerably moreactive than their 2β-substituted counterparts with the equatoriallyoriented 1α-hydroxy group. Thus, 2α-methyl vitamins 12 and 18 proved tobe 100 and 10 times, respectively, more active than their corresponding2β-isomers 13 and 19 in the cultures of HL-60 in vitro, whereas in thecase of 2-hydroxymethyl derivatives (20, 22 versus 21, 23) thesedifferences were slightly smaller. Since vitamins with 2β-methylsubstituent (13, 19) and both 2-hydroxymethyl analogs in 20S-series (22,23) have selective activity profiles combining high potency in cellulardifferentiation, and lack of calcemic activity, such compounds arepotentially useful as therapeutic agents for the treatment of cancer.

These results indicate that variation of substituents on C-2 in theparent 19-nor-1α,25-dihydroxyvitamin D₃ can change completely (andselectively) the biological potency of the analogs. These resultssuggest that 2α-methyl-19-nor-20S-1,25-(OH)₂D₃ has preferential activityon bone, making it a candidate for treatment of bone disease.

EXAMPLE 3

Preparation of 20(S)-1α,25-Dihydroxy-2α- and20(S)-1α,25-Dihydroxy-2α-methyl-26,27-dihomo-19-norvitamin D₃ (36 and37). Reference is made to SCHEME IV.

20(S)-25-[(Triethylsilyl)oxy]-des-A,B-26,27-dihomocholestan-8-one (32).To a solution of 20(S)-25-hydroxy Grundmann's ketone analog 31(Tetrionics, Madison, Wis.; 18.5 mg, 0.06 mmol) in anhydrous CH₂Cl₂ (60μL) was added 2,6-lutidine (17.4 μL, 0.15 mmol) and triethylsilyltrifluoromethanesulfonate (20.3 μL, 0.09 mmol). The mixture was stirredat room temperature under argon for 1 h. Benzene was added and water,and the organic layer was separated, washed with sat. CuSO₄ and water,dried (MgSO₄) and evaporated. The oily residue was redissolved in hexaneand applied on a silica Sep-Pak cartridge (2 g). Elution with hexane (10mL) gave a small quantity of less polar compounds; further elution withhexane/ethyl acetate (9:1) provided the silylated ketone. Finalpurification was achieved by HPLC (10-mm×25-cm Zorbax-Sil column, 4mL/min) using hexane/ethyl acetate (95:5) solvent system. Pure protectedhydroxy ketone 32 (16.7 mg, 66%) was eluted at R_(V) 37 mL as acolorless oil: ¹H NMR (CDCl₃) δ 0.573 (6H, q, J=7.9 Hz, 3× SiCH₂), 0.639(3H, s, 18-H₃), 0.825 (6H, t, J=7.5 Hz, 26- and 27-CH₃), 0.861 (3H, d,J=6.1 Hz, 21-H₃), 0.949 (9H, t, J=7.9 Hz, 3× SiCH₂CH₃), 2.45 (1H, dd,J=11.4, 7.6 Hz, 14α-H).

20(S)-1α,25-Dihydroxy-2-methylene-26,27-dihomo-19-norvitamin D₃ (35). Toa solution of phosphine oxide 33 (9.1 mg, 15.6 μmol) in anhydrous THF(150 μL) at 0° C. was slowly added n-BuLi (2.5 M in hexanes, 7 μL, 17.5μmol) under argon with stirring. The solution turned deep orange. It wasstirred for 10 min at 0° C., then cooled to -78° C. and a precooled(−78° C.) solution of protected hydroxy ketone 32 (16.5 mg, 39.0 μmol)in anhydrous THF (300+100 μL) was slowly added. The mixture was stirredunder argon at −78° C. for 1.5 h and at 0° C. for 19 h. Water and ethylacetate were added, and the organic phase was washed with brine, dried(MgSO₄) and evaporated. The residue was dissolved in hexane and appliedon a silica Sep-Pak cartridge, and washed with hexane/ethyl acetate(99.7:0.3, 20 mL) to give slightly impure 19-norvitamin derivative 34(ca. 4 mg). The Sep-Pak was then washed with hexane/ethyl acetate (96:4,10 mL) to recover some unchanged C,D-ring ketone (contaminated with,14-isomer), and with ethyl acetate (10 mL) to recover diphenylphosphineoxide 33 (ca. 6 mg) that was subsequently purified by HPLC (10-mm×25-cmZorbax-Sil column, 4 mL/min) using hexane/2-propanol (9:1) solventsystem; pure compound 33 (5.1 mg) was eluted at R_(V) 36 mL. Theprotected vitamin 34 was further purified by HPLC (6.2-mm×25-cmZorbax-Sil column, 4 mL/min) using hexane/ethyl acetate (99.9:0.1)solvent system. Pure compound 34 (3.6 mg, 67% yield considering therecovery of unreacted 33) was eluted at R_(V) 19 mL as a colorless oil:UV (in hexane) )max 244.0, 252.5, 262.5 nm; ¹H NMR (CDCl₃) δ 0.026,0.048, 0.066, and 0.079 (each 3H, each s, 4× SiCH₃), 0.544 (3H, s,18-H₃), 0.570 (6H, q, J=7.9 Hz, 3× SiCH₂), 0.821 (6H, t, J=7.5 Hz, 26-and 27-CH₃), 0.849 (3H, d, J=6.7 Hz, 21-H₃), 0.864 and 0.896 (9H and 9H,each s, 2× Si-t-Bu), 0.946 (9H, t, J=7.9 Hz, 3× SiCH₂CH₃), 1.99 (2H, m),2.18 (1H, dd, J=12.6, 8.2 Hz, 4β-H), 2.34 (1H, dd, J=13.0, 2.9 Hz,100-H), 2.46 (1H, dd, J=12.6,4.3 Hz, 4α-H), 2.51 (1H, dd, J=13.0, 6.2Hz, 10α-H), 2.82 (1H, br d, J=12 Hz, 9β-H), 4.43 (2H, m, 1β- and 3α-H),4.92 and 4.97 (1H and 1H, each s, ═CH₂), 5.84 and 6.22 (1H and 1H, eachd, J=11.2 Hz, 7- and 6-H); MS m/z (relative intensity) 786 (M⁺, 15), 757(M⁺-Et, 22), 729 (M⁺-t-Bu, 5), 654 (100), 522 (15), 366 (43), 201 (31).

Protected vitamin 34 (3.5 mg) was dissolved in benzene (150 μL) and theresin (AG 50W-X4,40 mg; prewashed with methanol) in methanol (550 μL)was added. The mixture was stirred at room temperature under argon for14 h, diluted with ethyl acetate/ether (1:1, 4 mL) and decanted. Theresin was washed with ether (8 mL) and the combined organic phaseswashed with brine and saturated NaHCO₃, dried (MgSO₄) and evaporated.The residue was purified by HPLC (6.2-mm×25-cm Zorbax-Sil column, 4mL/min) using hexane/2-propanol (9:1) solvent system. Analytically pure2-methylene-19-norvitamin 35 (1.22 mg, 62%) was collected at R_(V) 21 mLas a white solid: UV (in EtOH) λ_(max) 243.5, 252.0, 262.0 nm; ¹H NMR(CDCl₃) δ 0.550 (3H, s, 18-H₃), 0.855 (3H, d, J=6.8 Hz, 21-H₃), 0.860(6H, t, J=7.5 Hz, 26- and 27-CH₃), 2.00 (3H, m), 2.30 (1H, dd, J=13.3,8.6 Hz, 10α-H), 2.33 (1H, dd, J=13.3, 6.3 Hz, 41-H), 2.58 (1H, dd,J=13.3, 3.9 Hz, 4α-H), 2.82 (1H, br d, J=12 Hz, 9β-H), 2.85 (1H, dd,J=13.3, 4.7 Hz, 10β-H), 4.48 (2H, m, 1β- and 3α-H), 5.09 and 5.11 (1Hand 1H, each s, ═CH₂), 5.89 and 6.36 (1H and 1H, each d, J=11.3 Hz, 7-and 6-H); MS m/z (relative intensity) 444 (M⁺, 100), 426 (35), 408 (11),397 (19), 379 (32), 341 (31), 287 (32), 273 (43), 269 (28), 251 (22);exact mass calcd for C₂₉H₄₈O₃ 444.3603, found 444.3602.

20(S)-1α,25-Dihydroxy-2α- and20(S)-1α,25-Dihydroxy-2β-methyl-26,27-dihomo-19-norvitamin D₃ (36 and37). Tris(triphenylphosphine)rhodium (1) chloride (2.3 mg, 2.5 μmol) wasadded to dry benzene (2.5 mL) presaturated with hydrogen. The mixturewas stirred at room temperature until a homogeneous solution was formed(ca. 45 min). A solution of vitamin 35 (1.0 mg, 2.3 μmol) in dry benzene(0.5 mL) was then added and the reaction was allowed to proceed under acontinuous stream of hydrogen for 4.5 h. A new portion of the catalyst(2.3 mg, 2.5 μmol) was added and hydrogen was passed for additional 1 h.Benzene was removed under vacuum, the residue was redissolved inhexane/ethyl acetate (1:1, 2 mL) and applied on Waters silica Sep-Pak. Amixture of 2-methyl vitamins was eluted with the same solvent system (20mL). The compounds were further purified by HPLC (6.2 mm×25 cmZorbax-Sil column, 4 mL/min) using hexane/2-propanol (9:1) solventsystem. The mixture (ca. 1:1) of 2-methyl-19-norvitamins 36 and 37 (0.37mg, 37%) gave a single peak at R_(V) 23 mL. Separation of both epimerswas achieved by reversed-phase HPLC (6.2-mm×25-cm Zorbax-ODS column, 2mL/min) using methanol/water (85:15) solvent system. 20-Methyl vitamin37 was collected at R_(V) 21 mL and its 2α-epimer 36 at R_(V) 27 mL.

36: UV (in EtOH) λ_(max) 242.5, 251.0, 261.0; ¹H NMR (CDCl₃) δ 0.534(3H, s, 18-H₃), 0.851 (3H, d, J˜7 Hz, 21-H₃), 0.858 (6H, t, J=7.5 Hz,26- and 27-CH₃), 1.133 (3H, d, J=6.9 Hz, 2α-CH₃), 2.13 (1H, ˜t, J˜12 Hz,40-H), 2.23 (1H, br d, J=13.4 Hz, 10β-H), 2.60 (1H, dd, J=13.1, 4.4 Hz,4α-H), 2.80 (2H, m, 9,- and 100α-H), 3.61 (1H, m, w/2=26 Hz, 3α-H), 3.96(1H, m, w/2=13 Hz, 1β-H), 5.82 and 6.37 (1H and 1H, each d, J=11.2 Hz,7- and 6-H); MS m/z (relative intensity) 446 (M⁺, 53), 428 (46), 410(12), 399 (35), 381 (17), 289 (35), 273 (48), 271 (30), 253 (24), 69(100); exact mass calcd for C₂₉H₅₀O₃ 446.3760, found 446.3758.

37: UV (in EtOH) λ_(max) 242.5, 251.0, 261.0 nm; ¹H NMR (CDCl₃) δ 0.546(3H, s, 18-H₃), 0.855 (3H, d, J=6.8 Hz, 21-H₃), 0.860 (6H, t, J=7.4 Hz,26- and 27-CH₃), 1.143 (3H, d, J=6.9 Hz, 2β-CH₃), 2.34 (1H, dd, J=13.7,3.3 Hz, 4,-H), 2.43 (1H, br d, J=13.7 Hz, 4α-H), 2.80 (1H, dd, J=12 and4 Hz, 9β-H), 3.08 (1H, dd, J=12.8, 4.1 Hz, 10β-H), 3.50 (1H, m, w/2=26Hz, 1β-H), 3.90 (1H, m, w/2=11 Hz, 3α-H), 5.87 and 6.26 (1H and 1H, eachd, J=11.2 Hz, 7- and 6-H); MS m/z (relative intensity) 446 (M⁺, 39) 428(46), 410 (12), 399 (30), 381 (17), 289 (37), 273 (50), 271 (31), 253(27), 69 (100); exact mass calcd for C₂₉H₅₀O₃ 446.3760, found 446.3740.

Biological Activity of20(S)-1α,25-Dihydroxy-2α-methyl-26,27-dihomo-19-nor-vitamin D₃ and20(S)-1α,25-Dihydroxy-2β-methyl-26,27-dihomo-19-norvitamin D₃ (36 and37)

Competitive binding of the analogs to the porcine intestinal receptorwas carried out by the method described by Dame et al Biochemistry 25,4523-4534, 1986).

The differentiation of HL-60 promyelocytic into monocytes was determinedas described by Ostrem et al (J. Biol. Chem. 262 14164-14171, 1987).

TABLE 6 VDR Binding Properties^(a) and HL-60 DifferentiatingActivities^(b) of 2- Substituted Analogs of20(S)-1α,25-Dihydroxy-26,27-dihomo- 19-norvitamin D₃ VDR Binding HL-60Differentiation Compd. ED₅₀ Binding ED₅₀ Activity Compound no. (M) ratio(M) ratio 1α,25-(OH)₂D₃ 8.7 × 10⁻¹⁰ 1 4.0 × 10⁻⁹ 12α-methyl-26,27-dihomo- 36 3.1 × 10⁻⁹ 3.6 6.0 × 10⁻¹¹ 0.0119-nor-20(S)-1α,25-(OH)₂D₃ 2β-methyl-26,27-dihomo- 37 4.8 × 10⁻⁹ 5.5 1.1× 10⁻¹⁰ 0.03 19-nor-20(S)-1α,25-(OH)₂D₃ ^(a)Competitive binding of1α,25-(OH)₂D₃ and the synthesized vitamin D analogs to the porcineintestinal vitamin D receptor. The experiments were carried out intriplicate on two different occasions. The ED₅₀ values are derived fromdose-response curves and represent the analog concentration required for50% displacement of the radiolabeled 1α,25-(OH)₂D₃ from the receptorprotein. Binding ratio is the ratio of the analog average # ED₅₀ to theED₅₀ for 1α,25-(OH)₂D₃. ^(b)Induction of differentiation of HL-60promyelocytes to monocytes by 1α,25-(OH)₂D₃ and the synthesized vitaminD analogs. Differentiation state was determined by measuring thepercentage of cells reducing nitro blue tetrazolium (NBT). Theexperiment was repeated three times. The values ED₅₀ are derived fromdose-response curves and represent the analog concentration capable ofinducing 50% maturation. Differentiation activity radio is the # ratioof the analog average ED₅₀ to the ED₅₀ for 1α,25-(OH)₂D₃.

TABLE 7 Support of Intestinal Calcium Transport and Bone CalciumMobilization by 2-Substituted Analogs of of 20(S)-1α,25-Dihydroxy-26,27-dihomo-19-norvitamin D₃ in Vitamin D-Deficient Ratson a Low-Calcium Diet^(a) Ca Transport Serum Ca Compd. Amount S/M (mean± (mean ± Compound no. (pmol) SEM) SEM) one (control) 0 2.7 ± 0.3^(b)4.7 ± 0.2^(b) 1α,25-(OH)₂D₃ 260 7.2 ± 0.6^(c) 5.6 ± 0.2^(c)2α-methyl-26, 36 32 5.8 ± 0.4^(d) ¹ 5.9 ± 0.2^(d) ¹ 27-dihomo-19-nor-20(S)-1α, 65 8.4 ± 0.8^(d) ² 9.3 ± 0.2^(d) ² 25-(OH)₂D₃ none(control) 0 3.6 ± 0.4^(b) 5.0 ± 0.1^(b) 1α,25-(OH)₂D₃ 260 5.0 ± 0.4^(c)6.3 ± 0.2^(c) 2β-methyl-26, 37 65 4.7 ± 0.6^(d) ¹ 5.0 ± 0.0^(d) ¹27-dihomo- 19-nor-20(S)-1α, 260 5.2 ± 0.6^(d) ² 9.9 ± 0.3^(d) ²25-(OH)₂D₃ ^(a)Weanling male rats were maintained on a 0.47% Ca diet for1 week and then switched to a low-calcium diet containing 0.02% Ca foran additional 3 weeks. During the last week, they were dosed daily withthe appropriate vitamin D compound for 7 consecutive days. All doseswere administered intraperitoneally in 0.1 ml propylene glycol/ethanol(95:5). Controls received the vehicle. Determinations were made 24 hafter the last dose. There were at least 6 rats per group. Statistical #analysis was done by Student's t-test. Statistical data: serosal/mucosal(S/M), panel 1, b from c, and d², p < 0.001, b from d¹, p = 0.001; panel2, b from c, d¹, and d², p < 0.05; serum calcium, panel 1, b from c andd¹, p < 0.05, b from d², p < 0.001; panel 2, b from c, p < 0.01, b fromd¹, NS, b from d², p < 0.001.

EXAMPLE 4

Preparation of20(S)-1α,25-Dihydroxy-26,27-dimethylene-2α-methyl-19-norvitamin D₃ and20(S)-1α,25-Dihydroxy-26,27-dimethylene-2β-methyl-19-norvitamin D₃ (48and 49). Reference is made to SCHEMES V and VI.

20(S)-25-[(Triethylsilyl)oxy]-des-A,B-26,27-dimethylene-cholestan-8-one(42). To a solution of 20(8)-25-hydroxy Grundmann's ketone analog 41(Tetrionics, Madison, WI; 15.0 mg, 0.049 mmol) in anhydrous CH₂Cl₂ (50μL) was added 2,6-lutidine (15 μL, 0.129 mmol) and triethylsilyltrifluoromethanesulfonate (17.0 μL, 0.075 mmol). The mixture was stirredat room temperature under argon for 1 h. Benzene was added and water,and the organic layer was separated, washed with sat. CuSO₄ and water,dried (MgSO₄) and evaporated. The oily residue was redissolved in hexaneand applied on a silica Sep-Pak cartridge (2 g). Elution with hexane (10mL) gave a small quantity of less polar compounds; further elution withhexane/ethyl acetate (9:1) provided the silylated ketone. Finalpurification was achieved by HPLC (10-mm×25-cm Zorbax-Sil column, 4mL/min) using hexane/ethyl acetate (95:5) solvent system. Pure protectedhydroxy ketone 42 (9.4 mg, 46%) was eluted at R_(V) 39 mL as a colorlessoil: ¹H NMR (CDCl₃) δ 0.576 (6H, q, J=7.9 Hz, 3× SiCH₂), 0.638 (3H, s,18-H₃), 0.865 (3H, d, J=6.1 Hz, 21-H₃), 0.949 (9H, t, J=7.9 Hz, 3×SiCH₂CH₃), 2.45 (1H, dd, J=11.4, 7.5 Hz, 14α-H).20(S)-1α,25-Dihydroxy-26,27-dimethylene-2-methylene-19-norvitamin D3(47). To a solution of phosphine oxide 43 (17.7 mg, 30.4 μmol) inanhydrous THF (300 μL) at 0° C. was slowly added n-BuLi (2.5 M inhexanes, 13 μL, 32.5 μmol) under argon with stirring. The solutionturned deep orange. It was stirred for 10 mm at 0° C., then cooled to−78° C. and a precooled (−78° C.) solution of protected hydroxy ketone42 (17.8 mg, 42.3 μmol) in anhydrous THF (300+100 μL) was slowly added.The mixture was stirred under argon at −78° C. for 1.5 h and at 0° C.for 18 h. Water and ethyl acetate were added, and the organic phase waswashed with brine, dried (MgSO₄) and evaporated. The residue wasdissolved in hexane and applied on a silica Sep-Pak cartridge, andwashed with hexane/ethyl acetate (99.7:0.3, 20 mL) to give slightlyimpure 19-norvitamin derivative 44 (ca. 11 mg). The Sep-Pak was thenwashed with hexane/ethyl acetate (96:4, 10 mL) to recover some unchangedC,D-ring ketone (contaminated with 14β-isomer), and with ethyl acetate(10 mL) to recover diphenylphosphine oxide 43 (ca. 8 mg) that wassubsequently purified by HPLC (10-mm×25-cm Zorbax-Sil column, 4 mL/min)using hexane/2-propanol (9:1) solvent system; pure compound 43 (7.6 mg)was eluted at RV 36 mL. The protected vitamin 44 was further purified byHPLC (6.2-mm×25-cm Zorbax-Sil column, 4 mL/min) using hexane/ethylacetate (99.9:0.1) solvent system. Pure compound 44 (10.1 mg, 74% yieldconsidering the recovery of unreacted 43) was eluted at R_(V) 27 mL as acolorless oil: UV (in hexane) λ_(max) 244.0, 252.5, 262.5 nm; 1H NMR(CDCl₃) δ 0.027, 0.048, 0.067, and 0.080 (each 3H, each s, 4×SiCH₃),0.544 (3H, s, 18-H₃), 0.575 (6H, q, J=7.9 Hz, 3×SiCH₂), 0.854 (3H, d,J=6.1 Hz, 2t-H₃), 0.866 and 0.896 (9H and 9H, each s, 2×Si-t-Bu), 0.947(9H, t, J=7.9 Hz, 3×SiCH₂CH₃), 1.99 (2H, in), 2.18 (1H, dd, J=12.8, 8.6Hz, 4β-H), 2.34 (1H, dd, J=13.2, 2.7 Hz, 10β-H), 2.46 (1H, dd, J=12.8,4.4 Hz, 4α-H), 2.51 (1H, dd, J=13.2, 6.0 Hz, 10-H), 2.82 (1H, br d, J=12Hz, 9β-H), 4.42 (2H, m, 1β- and 3α-H), 4.92 and 4.97 (1H and 1H, each s,═CH₂), 5.84 and 6.22 (1H and 1H, each d, J=11.2 Hz, 7- and 6-H); MS m/z(relative intensity) 784 (M⁺, 8), 755 (M⁺-Et, 4), 727 (M⁺-t-Bu, 6), 652(100), 520 (31), 366 (49), 199 (23).

Protected vitamin 44 (7.0 mg) was dissolved in benzene (220 μL) and theresin (AG 50W-X4, 95 mg; prewashed with methanol) in methanol (1.2 mL)was added. The mixture was stirred at room temperature under argon for21 h, diluted with ethyl acetate/ether (1:1, 4 mL) and decanted. Theresin was washed with ether (10 mL) and the combined organic phaseswashed with brine and saturated NaHCO₃, dried (MgSO₄) and evaporated.The residue was separated by HPLC (6.2-mm×25-cm Zorbax-Sil column, 4mL/min) using hexane/2-propanol (9:1) solvent system and the followinganalytically pure 2-methylene-19-norvitamins were isolated:1×-hydroxy-25-dehydrovitamin 45 (0.68 mg, 17%) was collected at R_(V) 13mL, 1α-hydroxy-25-methoxyvitamin 46 (0.76 mg, 19%) was collected atR_(V) 16 mL and 1α,25-dihydroxyvitamin 47 (2.0 mg, 51%) was collected atR_(V) 21 mL.

45: WV (in EtOH) λ_(max) 243.5, 251.5, 262.0 nm; ¹H NMR (CDCl₃) δ 0.542(3H, s, 18-H₃), 0.847 (3H, d, J 6.5 Hz, 21-H₃), 1.93-2.07 (4H, m),2.18-2.25 (2H, m), 2.26-2.36 (4H, m), 2.58 (1H, dd, J=13.3, 3.9 Hz,4α-H), 2.82 (1H, br d, J=13 Hz, 9β-H), 2.85 (1H, dd, J=13.3, 4.5 Hz,10β-H), 4.48 (2H, m, 1β- and 3α-H), 5.09 and 5.11 (1H and 1H, each s,═CH₂), 5.32 (1H, m, w/2=7 Hz, 24-H), 5.88 and 6.36 (1H and 1H, each d,J=11.1 Hz, 7- and 6-H); MS m/z (relative intensity) 424 (M⁺, 100), 406(7), 339 (16), 287 (16), 271 (24), 269 (17), 251 (12); exact mass calcdfor C₂₉H₄₄O₂ 424.3341, found 424.3343. 46: UV (in EtOH) λ_(max) 243.5,252.0, 262.0 nm; ¹H NMR (CDCl₃) δ 0.553 (3H, s, 18-H₃), 0.858 (3H, d,J=6.5 Hz, 21-H₃), 1.95-2.05 (2H, m), 2.30 (1H, dd, J=13.3, 8.3 Hz,10α-H), 2.33 (1H, dd, 3=13.4, 6.0 Hz, 4β-H), 2.58 (1H, dd, J=13.4, 3.8Hz, 4α-H), 2.82 (1H, br d, J=13 Hz, 9β-H), 2.85 (1H, dd, J=13.3, 4.4 Hz,10β-H), 3.13 (3H, s, OCH₃), 4.48 (2H, m, 1β- and 3α-H), 5.09 and 5.11(1H and 1H, each s, ═CH₂), 5.89 and 6.36 (1H and 1H, each d, J=11.2 Hz,7- and 6-H); MS m/z (relative intensity) 456 (M⁺, 54), 424 (27), 406(12), 339 (16), 287 (13), 271 (41), 99 (100); exact mass calcd forC₃₀H₄₈O₃ 456.3603, found 456.3603.

47: UV (in EtOH) λ_(max) 243.5, 252.0, 262.0 nm; ¹H NMR (CDCl₃) δ 0.551(3H, s, 18-H₃), 0.859 (3H, d, J=6.6 Hz, 21-H₃), 1.95-2.05 (2H, m), 2.30(1H, dd, J=13.5, 8.4 Hz, 10α-H), 2.33 (1H, dd, J=13.3, 6.3 Hz, 4β-H),2.58 (1H, dd, J=13.3, 4.0 Hz, 4α-H), 2.82 (1H, br d, J=12 Hz, 9β-H),2.85 (1H, dd, J=13.5, 4.4 Hz, 10β-H), 4.48 (2H, m, 1β- and 3α-H), 5.09and 5.11 (1H and 1H, each s, ═CH₂), 5.89 and 6.36 (1H and 1H, each d,J=11.3 Hz, 7- and 6-H); MS m/z (relative intensity) 442 (M⁺, 100), 424(47), 406 (15), 339 (34), 287 (27), 271 (42), 269 (36), 251 (26); exactmass calcd for C₂₉H₄₆O₃ 442.3447, found 442.3442.

20(S)-1α,25-Dihydroxy-26,27-dimethylene-2α- and20(S)-1α,25-Dihydroxy-26,27-dimethylene-21β-methyl-19-norvitamin D₃ (48and 49). Tris(triphenylphosphine)rhodium (I) chloride (2.3 mg, 2.5 μmol)was added to dry benzene (2.5 mL) presaturated with hydrogen. Themixture was stirred at room temperature until a homogeneous solution wasformed (ca. 45 min). A solution of vitamin 47 (1.0 mg, 2.3 μmol) in drybenzene (0.5 mL) was then added and the reaction was allowed to proceedunder a continuous stream of hydrogen for 3 h. A new portion of thecatalyst (2.3 mg, 2.5 μmol) was added and hydrogen was passed foradditional 2 h. Benzene was removed under vacuum, the residue wasredissolved in hexane/ethyl acetate (1:1, 2 mL) and applied on Waterssilica Sep-Pak. A mixture of 2-methyl vitamins was eluted with the samesolvent system (20 mL). The compounds were further purified by HPLC (6.2mm×25 cm Zorbax-Sil column, 4 mL/min) using hexane/2-propanol (9:1)solvent system. The mixture (ca. 1:1) of 2-methyl-19-norvitamins 48 and49 (0.23 mg, 23%) gave a single peak at R_(V) 23 mL. Separation of bothepimers was achieved by reversed-phase HPLC (6.2-mm×25-cm Zorbax-ODScolumn, 2 mL/min) using methanol/water (85:15) solvent system. 2β-Methylvitamin 49 was collected at R_(V) 19 mL and its 2α-epimer 48 at R_(V) 24mL.

48: UV (in EtOH) λ_(max) 242.5, 251.0, 261.5 nm; ¹H NMR (CDCl₃) δ 0.534(3H, s, 18-H₃), 0.853 (3H, d, J=6.6 Hz, 21-H₃), 1.134 (3H, d, J=6.8 Hz,2α-CH₃), 2.13 (1H, ˜t, J˜12 Hz, 4β-H),2.22(1H, br d, J=13 Hz, 10β-H),2.60(1H, dd, J=12.8, 4.6 Hz, 4α-H), 2.80 (2H, m, 9β- and 10α-H), 3.61(1H, m, w/2=23 Hz, 3α-H), 3.96 (1H, m, w/2=11 Hz, 1β-H), 5.82 and 6.37(1H and 1H, each d, J=11.3 Hz, 7- and 6-H); MS m/z (relative intensity)444 (M⁺, 84), 426 (53), 289 (36), 271 (58), 253 (19); exact mass calcdfor C₂₉H₄₈O₃ 444.3603, found 444.3602.

49: UV (in EtOH) λ_(max) 242.5, 251.0, 261.5 nm; ¹H NMR (CDCl₃) δ 0.547(3H, s, 18-H₃), 0.859 (3H, d, J=6.8 Hz, 21-H₃), 1.143 (3H, d, J=6.8 Hz,2β-CH₃), 2.34 (1H, dd, J=13.7,-3.3 Hz, 4β-H), 2.43 (1H, br d, J=13.7 Hz,4α-H), 2.80 (1H, br d, J=12 Hz, 9β-H), 3.08 (1H, dd, J=12.9, 4.4 Hz,10β-H), 3.50 (1H, m, w/2=25 Hz, 1β-H), 3.90 (1H, m, w/2=12 Hz, 3α-H),5.87 and 6.26 (1H and 1H, each d, J=11.3 Hz, 7- and 6-H); MS m/z(relative intensity) 444 (M⁺, 75), 426 (59), 289 (34), 271 (59), 253(18); exact mass calcd for C₂₉H₁₈O₃ 444.3603, found 444.3611.

Biological Activity of20(S)-1α,25-Dihydroxy-26,27-dimethylene-2α-methyl-19-norvitamin D₃ and20(S)-1α,25-Dihydroxy-26,27-dimethylene-2β-methyl-19-norvitamin D₃ (48and 49)

Competitive binding of the analogs to the porcine intestinal receptorwas carried out by the method described by Dame et al (Biochemistry 2,4523-4534, 1986).

The differentiation of HL-60 promyelocytic into monocytes was determinedas described by Ostrem et al (J. Biol. Chem. 262, 14164-14171, 1987).

TABLE 8 VDR Binding Properties^(a) and HL-60 DifferentiatingActivities^(b) of 2-Substituted Analogs of 20(S)-1α,25-Dihydroxy-26,27-dimethylene-19-norvitamin D₃ VDR Binding HL-60 Differentiation Compd.ED₅₀ Binding ED₅₀ Activity Compound no. (M) ratio (M) ratio1α,25-(OH)₂D₃ 8.7 × 10⁻¹⁰ 1 4.0 × 10⁻⁹  1 2α-methyl- 48 3.5 × 10⁻⁹  4.04.4 × 10⁻¹¹ 0.01 26,27-dim- ethylene-19- nor-20(S)-1α, 25-(OH)₂D₃2β-methyl- 49 2.3 × 10⁻⁹  2.6 3.2 × 10⁻¹⁰ 0.08 26,27-dim- ethylene-19-nor-20(S)-1α, 25-(OH)₂D₃ ^(a)Competitive binding of 1α,25-(OH)₂D₃ andthe synthesized vitamin D analogs to the porcine intestinal vitamin Dreceptor. The experiments were carried out in triplicate on twodifferent occasions. The ED₅₀ values are derived from dose-responsecurves and represent the analog concentration required for 50%displacement of the radiolabeled 1α,25-(OH)₂D₃ from the receptorprotein. Binding ratio is the ratio of the analog average ED₅₀ to the #ED₅₀ for 1α,25-(OH)₂D₃. ^(b)Induction of differentiation of HL-60promyelocytes to monocytes by 1α,25-(OH)₂D₃ and the synthesized vitaminD analogs. Differentiation state was determined by measuring thepercentage of cells reducing nitro blue tetrazolium (NBT). Theexperiment was repeated three times. The values ED₅₀ are derived fromdose-response curves and represent the analog concentration capable ofinducing 50% maturation. Differentiation activity ratio is the ratio ofthe analog average # ED₅₀ to the ED₅₀ for 1α,25-(OH)₂D₃.

TABLE 9 Support of Intestinal Calcium Transport and Bone CalciumMobilization by 2-Substituted Analogs of of20(1S)-1α,25-Dihydroxy-26,27- dimethylene-19-norvitamin D₃ in VitaminD-Deficient Rats on a Low-Calcium Diet^(a) Compd. Amount Ca TransportS/M Serum Ca Compound no. (pmol) (mean ± SEM) (mean ± SEM) none(control)  0 2.7 ± 0.3^(b) 4.7 ± 0.2^(b) 1α,25-(OH)₂D₃ 260 7.2 ± 0.6^(c)5.6 ± 0.2^(c) 2α-methyl-26,27- 48  32 7.9 ± 1.0^(d) ¹ 6.9 ± 0.5^(d) ¹dimethylene- 19-nor-20(S)-1α,  65 9.0 ± 1.0^(d) ² 9.0 ± 0.3^(d) ²25-(OH)₂D₃ none (control)  0 3.6 ± 0.4^(b) 5.0 ± 0.1^(b) 1α,25-(OH)₂D₃260 5.0 ± 0.4^(c) 6.3 ± 0.2^(c) 2β-methyl-26,27- 49  65 4.9 ± 0.6^(d) ¹5.8 ± 0.2^(d) ¹ dimethylene- 19-nor-20(S)-1α, 260 5.4 ± 0.7^(d) ² 9.5 ±0.1^(d) ² 25-(OH)₂D₃ ^(a)Weanling male rats were maintained on a 0.47%Ca diet for 1 week and then switched to a low-calcium diet containing0.02% Ca for an additional 3 weeks. During the last week, they weredosed daily with the appropriate vitamin D compound for 7 consecutivedays. All doses were administered intraperitoneally in 0.1 ml propyleneglycol/ethanol (95:5). Controls received the vehicle. Determinationswere made 24 h after the last dose. There were at least 6 rats pergroup. Statistical analysis was done # by Student's t-test. Statisticaldata: serosal/mucosal (S/M), panel 1 b from c, d¹, and d², p < 0.001;panel 2, b from c, d¹, and d², p < 0.05; serum calcium, panel 1, b fromc, d¹, p < 0.05, b from d², p < 0.001; panel 2, b from c and d¹, p <0.01, b from d², p < 0.001.

For treatment purposes, the novel compounds of this invention defined beformula I may be formulated for pharmaceutical applications as asolution in innocuous solvents, or as an emulsion, suspension ordispersion in suitable solvents or carriers, or as pills, tablets orcapsules, together with solid carriers, according to conventionalmethods known in the art. Anby such formulations may also contain otherpahrmaceutically-acceptable acceptable and non-toxic excipients such asstabilizers, anti-oxidants, binders, coloring agents or emulsifying ortaste-modifying agents.

The compounds may be administered orally, topically, parenterally,sublingually, intranasally, or transdermally. The compounds areadvantageously administered by injection or by intravenous infusion orsuitable sterile solutions, or in the form of liquid or solid doses viathe alimentary canal, or in the form of creams, ointments, patches, orsimilar vehicles suitable for transdermal applications. Doses of from0.1 g to 50 kg per day of the compounds are appropriate for treatmentpurposes, such doses being adjusted according to the disease to betreated, its severity and the response of the subject as is wellunderstood in the art. Since the new compounds exhibit specificity ofaction, each may be suitably administered alone, or together with gradeddoses of another active vitamin D compound—e.g. 1α-hydroxyvitamin D₂ orD₃, or 1α,25-dihydroxyvitamin D₃—in situations where different degreesof bone mineral mobilization and calcium transport stimulation is foundto be advantageous.

Compositions for use in the above-mentioned treatment of psoriasis andother malignancies comprise an effective amount of one or more2-substituted-19-nor-vitamin D compound as defined by the above formulaI as the active ingredient, and a suitable carrier. An effective amountof such compounds for use in accordance with this invention is fromabout 0.01 μg to about 100 μg per gm of composition, and may beadministered topically, transdermally, orally, sublingually,intranasally, or parenterally in dosages of from about 0.1 μg/day toabout 100 μg/day.

The compounds may be formulated as creams, lotions, ointments, topicalpatches, pills, capsules or tablets, or in liquid form as solutions,emulsions, dispersions, or suspensions in pharmaceutically innocuous andacceptable solvent or oils, and such preparations may contain inaddition other pharmaceutically innocuous or beneficial components, suchas stabilizers, antioxidants, emulsifiers, coloring agents, binders ortaste-modifying agents.

The compounds are advantageously administered in amounts sufficient toeffect the differentiation of promyelocytes to normal macrophages.Dosages as described above are suitable, it being understood that theamounts given are to be adjusted in accordance with the severity of thedisease, and the condition and response of the subject as is wellunderstood in the art.

The formulations of the present invention comprise an active ingredientin association with a pharmaceutically acceptable carrier therefore andoptionally other therapeutic ingredients. The carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulations and not deleterious to the recipient thereof.

Formulations of the present invention suitable for oral administrationmay be in the form of discrete units as capsules, sachets, tablets orlozenges, each containing a predetermined amount of the activeingredient; in the form of a powder or granules; in the form of asolution or a suspension in an aqueous liquid or non-aqueous liquid; orin the form of an oil-in-water emulsion or a water-in-oil emulsion.

Formulations for rectal administration may be in the form of asuppository incorporating the active ingredient and carrier such ascocoa butter, or in the form of an enema.

Formulations suitable for parenteral administration convenientlycomprise a sterile oily or aqueous preparation of the active ingredientwhich is preferably isotonic with the blood of the recipient.

Formulations suitable for topical administration include liquid orsemi-liquid preparations such as liniments, lotions, applicants,oil-in-water or water-in-oil emulsions such as creams, ointments orpastes; or solutions or suspensions such as drops; or as sprays.

For asthma treatment, inhalation of powder, self-propelling or sprayformulations, dispensed with a spray can, a nebulizer or an atomizer canbe used. The formulations, when dispensed, preferably have a particlesize in the range of 10 to 100μ.

The formulations may conveniently be presented in dosage unit form andmay be prepared by any of the methods well known in the art of pharmacy.By the term “dosage unit” is meant a unitary, i.e. a single dose whichis capable of being administered to a patient as a physically andchemically stable unit dose comprising either the active ingredient assuch or a mixture of it with solid or liquid pharmaceutical diluents orcarriers.

In its broadest application, the present invention relates to any19-nor-2-alkyl analogs of vitamin D which have the vitamin D nucleus. Byvitamin D nucleus, it is meant a central part consisting of asubstituted chain of five carbon atoms which correspond to positions 8,14, 13, 17 and 20 of vitamin D, and at the ends of which are connectedat position 20 a structural moiety representing any of the typical sidechains known for vitamin D type compounds (such as R as previouslydefined herein), and at position 8 the 5,7-diene moiety connected to theA-ring of an active 1α-hydroxy vitamin D analog (as illustrated byformula I herein). Thus, various known modifications to the six-memberedC-ring and the five-membered D-ring typically present in vitamin D, suchas the lack of one or the other or both, are also embraced by thepresent invention.

Accordingly, compounds of the following formulae Ia, are along withthose of formula I, also encompassed by the present invention:

In the above formula Ia, the definitions of Y₁, Y₂, R₆, and Z are aspreviously set forth herein. With respect to X₁, X₂, X₃, X₄, X₅, X₆, X₇,X₈ and X₉, these substituents may be the same or different and areselected from hydrogen or lower alkyl, i.e. a C₁₋₅ alkyl such as methyl,ethyl or n-propyl. In addition, paired substituents X₁ and X₄ or X₅, X₂or X₃ and X₆ or X₇; X₄ or X₅ and X₈ or X₉, when taken together with thethree adjacent carbon atoms of the central part of the compound, whichcorrespond to positions 8, 14, 13 or 14,, 13, 17 or 13, 17, 20respectively, can be the same or different and form a saturated orunsaturated, substituted or unsubstituted, carbocyclic 3, 4, 5, 6 or 7membered ring.

Preferred compounds of the present invention may be represented by oneof the following formulae:

In the above formulae Ib, Ic, Id, Ie, If, Ig and Ih, the definitions ofY₁, Y₂, R₆, R, Z, X₁, X₂, X₃, X₄, X₅, X₆, X₇ and X₈ fare as previouslyset forth herein. The substituent Q represents a saturated orunsaturated, substituted or unsubstituted, hydrocarbon chain comprisedof 0, 1, 2, 3 or 4 carbon atoms, but is preferably the group —(CH₂)_(k)—where k is an integer equal to 2 or 3.

Methods for making compounds of formulae Ia-Ih are known. Specifically,reference is made to International Application Number PCT/FP94/02294filed Jul. 7, 1994 and published Jan. 19, 1995 under InternationalPublication Number WO95/01960.

I claim:
 1. A method of treating metabolic bone disease where it isdesired to maintain or increase bone mass comprising administering to apatient with said disease an effective amount of a compound selectedfrom the group consisting of19-nor-26,27-dihomo-20(S)-2α-methyl-1α,25-dihydroxyvitamin D₃,19-nor-26,27-dihomo-20(S)-2β-methyl-1α,25-dihydroxyvitamin D₃,19-nor-26,27-dimethylene-20(S)-2α-methyl-1α,25-dihydroxyvitamin D₃, and19-nor-26,27-dimethylene-20(S)-2β-methyl-1α,25-dihydroxyvitamin D₃. 2.The method of claim 1 where the disease is senile osteoporosis.
 3. Themethod of claim 1 where the disease is postmenopausal osteoporosis. 4.The method of claim 1 where the disease is steroid-induced osteoporosis.5. The method of claim 1 where the disease is low bone turnoverosteoporosis.
 6. The method of claim 1 where the disease isosteomalacia.
 7. The method of claim 1 where the disease is renalosteodystrophy.
 8. The method of claim 1 wherein the compound isadministered orally.
 9. The method of claim 1 wherein the compound isadministered parenterally.
 10. The method of claim 1 wherein thecompound is administered transdermally.
 11. The method of claim 1wherein the compound is administered in a dosage of from 0.1 μg to 50 μgper day.